WorldWideScience

Sample records for optimised electroporation mediated

  1. Optimised electroporation mediated DNA vaccination for treatment of prostate cancer.

    LENUS (Irish Health Repository)

    Ahmad, Sarfraz

    2010-01-01

    , preventing tumour occurrence in 54% of treated animals. Vaccination with phPSA resulted in anti-hPSA Abs production and a significant production of IFNgamma was observed in immunised animals (p < 0.05). Immune responses were tumour specific and were transferable in adoptive T cell transfer experiments. CONCLUSIONS: This phPSA plasmid electroporation vaccination strategy can effectively activate tumour specific immune responses. Optimisation of the approach indicated that a four-dose regimen provided highest tumour protection. In vivo electroporation mediated vaccination is a safe and effective modality for the treatment of prostate cancer and has a potential to be used as a neo-adjuvant or adjuvant therapy.

  2. Electroporation-mediated gene delivery.

    Science.gov (United States)

    Young, Jennifer L; Dean, David A

    2015-01-01

    Electroporation has been used extensively to transfer DNA to bacteria, yeast, and mammalian cells in culture for the past 30 years. Over this time, numerous advances have been made, from using fields to facilitate cell fusion, delivery of chemotherapeutic drugs to cells and tissues, and most importantly, gene and drug delivery in living tissues from rodents to man. Electroporation uses electrical fields to transiently destabilize the membrane allowing the entry of normally impermeable macromolecules into the cytoplasm. Surprisingly, at the appropriate field strengths, the application of these fields to tissues results in little, if any, damage or trauma. Indeed, electroporation has even been used successfully in human trials for gene delivery for the treatment of tumors and for vaccine development. Electroporation can lead to between 100 and 1000-fold increases in gene delivery and expression and can also increase both the distribution of cells taking up and expressing the DNA as well as the absolute amount of gene product per cell (likely due to increased delivery of plasmids into each cell). Effective electroporation depends on electric field parameters, electrode design, the tissues and cells being targeted, and the plasmids that are being transferred themselves. Most importantly, there is no single combination of these variables that leads to greatest efficacy in every situation; optimization is required in every new setting. Electroporation-mediated in vivo gene delivery has proven highly effective in vaccine production, transgene expression, enzyme replacement, and control of a variety of cancers. Almost any tissue can be targeted with electroporation, including muscle, skin, heart, liver, lung, and vasculature. This chapter will provide an overview of the theory of electroporation for the delivery of DNA both in individual cells and in tissues and its application for in vivo gene delivery in a number of animal models.

  3. Short-fragment DNA-mediated in vivo DNA electroporation delivery.

    Science.gov (United States)

    Peng, Jinliang; Zhao, Yonggang; Xu, Yuhong

    2014-01-01

    Electroporation is an effective physical delivery method. A variety of factors have been shown to affect the electroporation-mediated gene delivery efficiency. Here we report the usefulness of noncoding short-fragment DNA (sf-DNA) for facilitating electroporation-mediated gene transfer. The plasmid pGL3-control encoding firefly luciferase was injected into tissue together with or without sf-DNA in different length or dose. Immediately after injection, the tissues were electroporated and the level of luciferase activity was assessed 24 h later. The results showed that plasmid DNA formulated with sf-DNA resulted in significant improvement in electroporation-mediated gene transfer efficiency. The effect is dose and length dependent, and also found in low-voltage electroporation. These results indicated that sf-DNA can be used as a helper molecule to improve the electroporation-mediated gene transfection efficiency.

  4. Electroporation-mediated gene transfer directly to the swine heart.

    Science.gov (United States)

    Hargrave, B; Downey, H; Strange, R; Murray, L; Cinnamond, C; Lundberg, C; Israel, A; Chen, Y-J; Marshall, W; Heller, R

    2013-02-01

    In vivo gene transfer to the ischemic heart via electroporation holds promise as a potential therapeutic approach for the treatment of heart disease. In the current study, we investigated the use of in vivo electroporation for gene transfer using three different penetrating electrodes and one non-penetrating electrode. The hearts of adult male swine were exposed through a sternotomy. Eight electric pulses synchronized to the rising phase of the R wave of the electrocardiogram were administered at varying pulse widths and field strengths following an injection of either a plasmid encoding luciferase or one encoding green fluorescent protein. Four sites on the anterior wall of the left ventricle were treated. Animals were killed 48 h after injection and electroporation and gene expression was determined. Results were compared with sites in the heart that received plasmid injection but no electric pulses or were not treated. Gene expression was higher in all electroporated sites when compared with injection only sites demonstrating the robustness of this approach. Our results provide evidence that in vivo electroporation can be a safe and effective non-viral method for delivering genes to the heart, in vivo.

  5. Transdermal Delivery of Piroxicam by Surfactant Mediated Electroporation

    Institute of Scientific and Technical Information of China (English)

    ZAN Jia; JIANG Guoqiang; LIN Ying; TAN Fengping; DING Fuxin

    2005-01-01

    A lipophilic, nonsteroidal antiinflammation drug, piroxicam, was administered by skin electroporation using short, high-voltage pulses. The transdermal delivery of piroxicam during the electroporation was buffered due to the higher partition in skin lipids than in aqueous environments, which is called entrapment. Entrapment is the main resistance to transdermal delivery of lipophilic drugs. Two types of surfactants were used to enhance the skin electroporation. Tween 80 (0.2 g/L) and sodium dodecyl sulphate (SDS, 3 mg/mL) improve the solubility and diffusion rate of the drug in the hydrophobic local transport regions and reduce the entrapment of piroxicam in the skin. The transdermal delivery rate of piroxicam is increased 30- to 50-fold. However, the entrapment of piroxicam in the skin still occurred when Tween 80 was added. The SDS provides higher and more stable transdermal delivery rates of piroxicam than Tween 80, and also reduces the entrapment of piroxicam in the skin.

  6. The role of ion electrophoresis in electroporation-mediated molecular delivery

    Science.gov (United States)

    Li, Jianbo; Lin, Hao

    2009-11-01

    Electroporation is a widely applied technique to deliver active molecules into the cellular compartment, to perform a variety of tasks such as gene therapy and directed stem cell differentiation. In this technique, an electric field transiently permeabilizes the cellular membrane to facilitate molecular exchange. While the permeabilization process is relatively well-understood, the transport mechanisms for molecular delivery are still under debate. In this work, the role of ion electrophoresis in electroporation-mediated molecular delivery is investigated using numerical simulations. The result indicates that ion electrophoresis is the dominant mode of transport in the delivery of small charged molecules. Furthermore, the achievable intracellular concentration is strongly influenced by the conductivity difference between the cytoplasm and the buffer, a phenomenon known as ``field-amplified sample stacking''. The result agrees well with the fluorescence measurement by Gabriel and Teissi'e (1999), and suggests a new possibility to simultaneously improve cell viability and efficiency in electroporation-mediated molecular delivery.

  7. In vivo electroporation mediated gene delivery to the beating heart.

    Directory of Open Access Journals (Sweden)

    Erick L Ayuni

    Full Text Available Gene therapy may represent a promising alternative strategy for cardiac muscle regeneration. In vivo electroporation, a physical method of gene transfer, has recently evolved as an efficient method for gene transfer. In the current study, we investigated the efficiency and safety of a protocol involving in vivo electroporation for gene transfer to the beating heart. Adult male rats were anesthetised and the heart exposed through a left thoracotomy. Naked plasmid DNA was injected retrograde into the transiently occluded coronary sinus before the electric pulses were applied. Animals were sacrificed at specific time points and gene expression was detected. Results were compared to the group of animals where no electric pulses were applied. No post-procedure arrhythmia was observed. Left ventricular function was temporarily altered only in the group were high pulses were applied; CK-MB (Creatine kinase and TNT (Troponin T were also altered only in this group. Histology showed no signs of toxicity. Gene expression was highest at day one. Our results provide evidence that in vivo electroporation with an optimized protocol is a safe and effective tool for nonviral gene delivery to the beating heart. This method may be promising for clinical settings especially for perioperative gene delivery.

  8. Electroporation-mediated delivery of genes in rodent models of lung contusion.

    Science.gov (United States)

    Machado-Aranda, David; Raghavendran, Krishnan

    2014-01-01

    Several of the biological processes involved in the pathogenesis of acute lung injury and acute respiratory distress syndrome after lung contusion are regulated at a genetic and epigenetic level. Thus, strategies to manipulate gene expression in this context are highly desirable not only to elucidate the mechanisms involved but also to look for potential therapies. In the present chapter, we describe mouse and rat models of inducing blunt thoracic injury followed by electroporation-mediated gene delivery to the lung. Electroporation is a highly efficient and easily reproducible technique that allows circumvention of several of lung gene delivery challenges and safety issues present with other forms of lung gene therapy.

  9. Bystander Effect Induced by Electroporation is Possibly Mediated by Microvesicles and Dependent on Pulse Amplitude, Repetition Frequency and Cell Type.

    Science.gov (United States)

    Prevc, Ajda; Bedina Zavec, Apolonija; Cemazar, Maja; Kloboves-Prevodnik, Veronika; Stimac, Monika; Todorovic, Vesna; Strojan, Primoz; Sersa, Gregor

    2016-10-01

    Bystander effect, a known phenomenon in radiation biology, where irradiated cells release signals which cause damage to nearby, unirradiated cells, has not been explored in electroporated cells yet. Therefore, our aim was to determine whether bystander effect is present in electroporated melanoma cells in vitro, by determining viability of non-electroporated cells exposed to medium from electroporated cells and by the release of microvesicles as potential indicators of the bystander effect. Here, we demonstrated that electroporation of cells induces bystander effect: Cells exposed to electric pulses mediated their damage to the non-electroporated cells, thus decreasing cell viability. We have shown that shedding microvesicles may be one of the ways used by the cells to mediate the death signals to the neighboring cells. The murine melanoma B16F1 cell line was found to be more electrosensitive and thus more prone to bystander effect than the canine melanoma CMeC-1 cell line. In B16F1 cell line, bystander effect was present above the level of electropermeabilization of the cells, with the threshold at 800 V/cm. Furthermore, with increasing electric field intensities and the number of pulses, the bystander effect also increased. In conclusion, electroporation can induce bystander effect which may be mediated by microvesicles, and depends on pulse amplitude, repetition frequency and cell type.

  10. Electroporation-mediated Delivery of Genes in Rodent Models of Lung Contusion

    OpenAIRE

    2014-01-01

    Several of the biological processes involved in the pathogenesis of acute lung injury and acute respiratory distress syndrome after lung contusion are regulated at a genetic and epigenetic level. Thus, strategies to manipulate gene expression in this context are highly desirable not only to elucidate the mechanisms involved but also to look for potential therapies. In the present chapter, we describe mouse and rat models of inducing blunt thoracic injury followed by electroporation-mediated g...

  11. Transgenic expression of green fluorescent protein in caprine embryos produced through electroporation-aided sperm-mediated gene transfer.

    Science.gov (United States)

    Kumar Pramod, R; Kumar, Rakesh; Mitra, Abhijit

    2016-01-15

    Current methods of transgenic animal production are afflicted by low efficiency and high cost. Recently, the electroporation aided sperm-mediated gene transfer (SMGT) emerges as a promising alternative with variable success rate. Among the domestic animal species, the electroporation-aided SMGT is less investigated in goats, except a few reports in which attempts have been made using the auto-uptake method of SMGT. In this study, we report an optimized electroporation condition for SMGT of caprine sperm cells. Results of this study demonstrated that electroporation of caprine sperm cells at 300 V for 200 mS in TALP medium allowed the maximum uptake of foreign DNA with minimum adverse effects on the vital semen parameters viz., progressive motility, viability, and membrane and acrosome integrity. Further, DNA binding assay revealed DNA uptake by 81.3% sperm cells when 1.0 μg of DNA was used under optimum electroporation conditions as compared to 16.5% on simple incubation. The qPCR analysis showed four-fold more (Pelectroporation than incubation. A similar cleavage rate was observed after IVF using either electroporated (23.20 ± 1.20) or non-electroporated (25.20 ± 2.41) sperm cells suggesting the absence of adverse effect of electroporation on the fertilizing ability. Out of the 116 embryos produced by electroporated sperm, five (4.31%) embryos showed the expression of the foreign gene. In conclusion, our results confirm that using optimized electroporation conditions, the caprine sperm cells can uptake foreign DNA effectively with minimum negative effect on the semen parameters and could produce transgenic embryos.

  12. Optimisation of a 96-well electroporation assay for postnatal rat CNS neurons suitable for cost-effective medium-throughput screening of genes that promote neurite outgrowth

    Directory of Open Access Journals (Sweden)

    Thomas eHutson

    2011-12-01

    Full Text Available Following an injury, central nervous system (CNS neurons show a very limited regenerative response which results in their failure to successfully form functional connections with their original target. This is due in part to the reduced intrinsic growth state of CNS neurons, which is characterised by their failure to express key regeneration-associated genes (RAGs and by the presence of growth inhibitory molecules in CNS environment that form a molecular and physical barrier to regeneration. Here we have optimised a 96-well electroporation and neurite outgrowth assay for postnatal rat cerebellar granule neurons cultured upon an inhibitory cellular substrate expressing myelin-associated glycoprotein or a mixture of growth-inhibitory chondroitin sulphate proteoglycans. Optimal electroporation parameters resulted in 25% transfection efficiency and 50% viability for postnatal rat cerebellar granule neurons (CGNs. The neurite outgrowth of transduced neurons was quantitatively measured using a semi-automated image capture and analysis system. The neurite outgrowth was significantly reduced by the inhibitory substrates which we demonstrated could be partially reversed using a Rho Kinase inhibitor. We are now using this assay to screen large sets of RAGs for their ability to increase neurite outgrowth on a variety of growth inhibitory and permissive substrates.

  13. Stable Somatic Gene Expression in Mouse Lungs Following Electroporation-mediated Tol2 Transposon Delivery.

    Science.gov (United States)

    Muliawan, Hary Sakti; Nakayama, Kazuhiko; Yagi, Keiko; Ikeda, Koji; Yagita, Kazuhiro; Hirata, Ken-ichi; Emoto, Noriaki

    2015-10-07

    Gene delivery to the lung has rapidly progressed as an important method for studying various chronic lung diseases. Viral vectors, albeit highly efficient, are limited by the host immune response. Electroporation, a well-known non-viral method, can efficiently deliver genes to the lung, but is unable to induce stable gene expression. The Tol2 transposon is another non-viral method that can induce stable gene expression by reinserting its genes into the host genome. In this study, we combined electroporation and Tol2 transposons to obtain stable, high-level gene expression in the mouse lung. Tol2 transposon plasmids (pT2A-EGFP; Tol2, pCAGGS-TP; transposase) were optimized in vitro, and the electroporation procedure (pCAG-EGFP) was optimized in mouse lungs. After optimization, a combination of electroporation plus the Tol2 transposon was used in a comparative analysis with electroporation plus pCAG-EGFP. GFP expression levels were quantified and visualized on days 4 and 7 post-electroporation. We successfully reproduced the Tol2 transposon system in vitro and the electroporation procedure in vivo. We observed sustainable GFP expression using electroporation plus the Tol2 transposon on days 4 and 7, while electroporation plus pCAG-EGFP resulted in decreased GFP expression on day 7. We were able to induce high-level, stable gene expression in mouse lungs using a combination of electroporation and the Tol2 transposon. This represents a safer method for lung gene delivery that can be used as an alternative to viral vectors.

  14. OPTIMAL ELECTROPORATION CONDITION FOR SPERM MEDIATED GENE TRANSFER IN STRIPPED CATFISH (Pangasionodon hypophthalmus

    Directory of Open Access Journals (Sweden)

    Raden Roro Sri Pudji Sinarni Dewi

    2010-06-01

    Full Text Available The success of transgenic fish production has been achieved through eggs fertilization using electroporated sperms carrying exogenous DNA. This study was conducted in order to obtain the optimal electroporation condition for stripped catfish sperm. A plasmid containing green fluorescent protein (GFP gene driven by carp β-actin promoter was transferred into sperm using electrophoresis method towards transgenic stripped catfish (Pangasionodon hypophthalmus production. Electroporation was carried out using square wave shock with pulse length of 30 ms and pulse interval of 0.1 sec. Treatments are combination between voltage (50 V, 75 V, and 100 V and pulse number (1 and 3. Exogenous DNA concentration used was 10 μg/mL of Tris-EDTA. Results showed that increasing the voltage from 50 to 100 decreased sperm motility, while pulse number did not affect sperm motility. Voltage of 50 gave the best motility of sperm, although sperm viability relatively similar between treatments and control except at 100 V with 3 pulses number. Further, electroporation-treated sperms were able to fertilize eggs. Higher hatching rate of eggs was obtained in electroporation treatment at 50 V with pulse number of 1 and 3. The persistence of transferred GFP was detected in electroporated and incubated sperms (control. However, GFP was only detected in larvae from eggs that were fertilized by electroporated sperm. Thus, electroporation could be applied to produce transgenic stripped catfish.

  15. Factors influencing electroporation-mediated gene transfer to Stylosanthes guianensis (Aubl. Sw. protoplasts

    Directory of Open Access Journals (Sweden)

    Quecini V.M.

    2002-01-01

    Full Text Available In order to develop a high-efficiency and reproducible transformation protocol for Stylosanthes guianensis we assessed the biological and physical parameters affecting plant electroporation protoplasts. Energy input, as combinations of electric field strengths discharged by different capacitors, electroporation buffer and DNA form were evaluated. Transformation efficiency was assayed in vivo as transient reporter gene expression, using the GFP-coding gene mgfp5 driven by a CaMV 35S constitutive promoter. Energy input and electric field strength had a critical influence on transgene expression with higher transformation levels being achieved with 250 V.cm-1 discharged by 900 and 1000 muF capacitors. Linear plasmid DNA, the absence of chloride and the presence of calcium ions also increased transient gene expression, albeit not significantly.

  16. Electroporation mediated DNA vaccination directly to a mucosal surface results in improved immune responses

    OpenAIRE

    Kichaev, Gleb; Mendoza, Janess M; Amante, Dinah; Smith, Trevor RF; McCoy, Jay R; Sardesai, Niranjan Y.; Kate E. Broderick

    2013-01-01

    In vivo electroporation (EP) has been shown to be a highly efficient non-viral method for enhancing DNA vaccine delivery and immunogenicity, when the site of immunization is the skin or muscle of animals and humans. However, the route of entry for many microbial pathogens is via the mucosal surfaces of the human body. We have previously reported on minimally invasive, surface and contactless EP devices for enhanced DNA delivery to dermal tissue. Robust antibody responses were induced followin...

  17. Transport, resealing, and re-poration dynamics of two-pulse electroporation-mediated molecular delivery.

    Science.gov (United States)

    Demiryurek, Yasir; Nickaeen, Masoud; Zheng, Mingde; Yu, Miao; Zahn, Jeffrey D; Shreiber, David I; Lin, Hao; Shan, Jerry W

    2015-08-01

    Electroporation is of interest for many drug-delivery and gene-therapy applications. Prior studies have shown that a two-pulse-electroporation protocol consisting of a short-duration, high-voltage first pulse followed by a longer, low-voltage second pulse can increase delivery efficiency and preserve viability. In this work the effects of the field strength of the first and second pulses and the inter-pulse delay time on the delivery of two different-sized Fluorescein-Dextran (FD) conjugates are investigated. A series of two-pulse-electroporation experiments were performed on 3T3-mouse fibroblast cells, with an alternating-current first pulse to permeabilize the cell, followed by a direct-current second pulse. The protocols were rationally designed to best separate the mechanisms of permeabilization and electrophoretic transport. The results showed that the delivery of FD varied strongly with the strength of the first pulse and the size of the target molecule. The delivered FD concentration also decreased linearly with the logarithm of the inter-pulse delay. The data indicate that membrane resealing after electropermeabilization occurs rapidly, but that a non-negligible fraction of the pores can be reopened by the second pulse for delay times on the order of hundreds of seconds. The role of the second pulse is hypothesized to be more than just electrophoresis, with a minimum threshold field strength required to reopen nano-sized pores or defects remaining from the first pulse. These results suggest that membrane electroporation, sealing, and re-poration is a complex process that has both short-term and long-term components, which may in part explain the wide variation in membrane-resealing times reported in the literature.

  18. Calcium Electroporation

    DEFF Research Database (Denmark)

    Frandsen, Stine Krog; Gibot, Laure; Madi, Moinecha;

    2015-01-01

    BACKGROUND: Calcium electroporation describes the use of high voltage electric pulses to introduce supraphysiological calcium concentrations into cells. This promising method is currently in clinical trial as an anti-cancer treatment. One very important issue is the relation between tumor cell kill...... efficacy-and normal cell sensitivity. METHODS: Using a 3D spheroid cell culture model we have tested the effect of calcium electroporation and electrochemotherapy using bleomycin on three different human cancer cell lines: a colorectal adenocarcinoma (HT29), a bladder transitional cell carcinoma (SW780......), and a breast adenocarcinoma (MDA-MB231), as well as on primary normal human dermal fibroblasts (HDF-n). RESULTS: The results showed a clear reduction in spheroid size in all three cancer cell spheroids three days after treatment with respectively calcium electroporation (p

  19. Imaging the dynamics of individual electropores

    OpenAIRE

    Sengel, Jason T.; Wallace, Mark I.

    2016-01-01

    Electroporation is a widely used technique to permeabilize cell membranes. Despite its prevalence, our understanding of the mechanism of voltage-mediated pore formation is incomplete; methods capable of visualizing the time-dependent behavior of individual electropores would help improve our understanding of this process. Here, using optical single-channel recording, we track multiple isolated electropores in real time in planar droplet interface bilayers. We observe individual, mobile defect...

  20. Electroporation of heterogeneous lipid membranes.

    Science.gov (United States)

    Reigada, Ramon

    2014-03-01

    Electroporation is the basis for the transfection of genetic material and for drug delivery to cells, including electrochemotherapy for cancer. By means of molecular dynamics many aspects of membrane electroporation have been unveiled at the molecular detail in simple, homogeneous, lipid bilayers. However, the correspondence of these findings \\with the process happening in cell membranes requires, at least, the consideration of laterally structured membranes. Here, I present a systematic molecular dynamics study of bilayers composed of different liquid-ordered and liquid-disordered lipid phases subjected to a transversal electric field. The simulations reveal two significant results. First, the electric field mainly affects the properties of the disordered phases, so that electroporation takes place in these membrane regions. Second, the smaller the disordered domains are, the faster they become electroporated. These findings may have a relevant significance in the experimental application of cell electroporation in vivo since it implies that electro-induced and pore-mediated transport processes occur in particularly small disordered domains of the plasma membrane, thus locally affecting only specific regions of the cell.

  1. Experimental study of electroporation-mediated plasmid gene expression in skin and incisional wound%电穿孔介导质粒基因在皮肤和线性伤口中的表达

    Institute of Scientific and Technical Information of China (English)

    高振; 宋楠; 武晓莉; 曾谊林; 刘伟

    2008-01-01

    Objective To explore the feasibility of electroporation mediated gone transfer in rat incisional wound.Methods 12 Sprague-Dawley rat's dorsal skins were electroporated(800 voltages in amplitude with 6 square wsve pulses,each lasting 20 milliseconds with 200 millisecond interval)after injection of plasmid DNA(1 μg/μl,in 100 μl PBS)containing enhanced green fluorescence protein(EGFP)gene.Electroporated skins were incisiomdly wounded 24 hours after electroporation.Specimens were harvested at day 2,4,6,14,then EGFP expression in dennis wag observed and quantitatively analyzed with integrated optical density(IOD)followed by H&E staimng.Results Eleetroporation can mediate EGFP expression in epidermis,dermis and pannicuhs muscle.The expression level in dermis Wag the highest at dav 2(IOD=3.50±1.45)and disappeared at day 14.EGFP expression Wag not found in dennis if no electroporation apphed after plasmid injection(IOD=0).Conclusion Electroporation can mediate plagmid gone expression in ineisional wound efficiently and widely.%目的 研究在线性伤口中进行电穿孔基因转染的可行性.方法 于12只Sprague-Dawley大鼠背部皮肤内注射增强型绿色荧光蛋白(EGFP)质粒(1 μg/μl)后,在注射局部进行电穿孔(800 V/cm,6个方波,每个持续20ms,间隔为200ms).电穿孔后24 h在电穿孔部位做线性切口,于第2、4、6、14天收集标本.观察EGFP表达并计算其真皮内积分光密度(IOD).然后进行HE染色.结果 电穿孔使EGFP在表皮、真皮以及肉膜内表达,真皮内表达量在第2天最高(IOD=3.50±1.45),至第14天消失,而单纯质粒注射真皮内无表达(IOD=0).结论 电穿孔能够介导质粒在线性伤口中高效而广泛地表达.

  2. Fluorescent protein tagging of endogenous protein in brain neurons using CRISPR/Cas9-mediated knock-in and in utero electroporation techniques

    Science.gov (United States)

    Uemura, Takeshi; Mori, Takuma; Kurihara, Taiga; Kawase, Shiori; Koike, Rie; Satoga, Michiru; Cao, Xueshan; Li, Xue; Yanagawa, Toru; Sakurai, Takayuki; Shindo, Takayuki; Tabuchi, Katsuhiko

    2016-01-01

    Genome editing is a powerful technique for studying gene functions. CRISPR/Cas9-mediated gene knock-in has recently been applied to various cells and organisms. Here, we successfully knocked in an EGFP coding sequence at the site immediately after the first ATG codon of the β-actin gene in neurons in the brain by the combined use of the CRISPR/Cas9 system and in utero electroporation technique, resulting in the expression of the EGFP-tagged β-actin protein in cortical layer 2/3 pyramidal neurons. We detected EGFP fluorescence signals in the soma and neurites of EGFP knock-in neurons. These signals were particularly abundant in the head of dendritic spines, corresponding to the localization of the endogenous β-actin protein. EGFP knock-in neurons showed no detectable changes in spine density and basic electrophysiological properties. In contrast, exogenously overexpressed EGFP-β-actin showed increased spine density and EPSC frequency, and changed resting membrane potential. Thus, our technique provides a potential tool to elucidate the localization of various endogenous proteins in neurons by epitope tagging without altering neuronal and synaptic functions. This technique can be also useful for introducing a specific mutation into genes to study the function of proteins and genomic elements in brain neurons. PMID:27782168

  3. Electroporation of adult zebrafish.

    Science.gov (United States)

    Rao, N Madhusudhana; Rambabu, K Murali; Rao, S Harinarayana

    2008-01-01

    We generated transient transgenic zebrafish by applying electrical pulses subsequent to injection of DNA into muscle tissue of 3-6-month old adult zebrafish. Electroporation parameters, such as number of pulses, voltage, and amount of plasmid DNA, were optimized and found that 6 pulses of 40 V/cm at 15 mug/fish increased the luciferase expression by 10-fold compared with those in controls. By measuring the expression of luciferase, in vivo by electroporation in adult zebrafish and in vitro using fish cell line (Xiphophorus xiphidium A2 cells), the strength of three promoters (CMV, human EF-1alpha, and Xenopus EF-1alpha) was compared. Subsequent to electroporation after injecting DNA in the mid region of zebrafish, expression of green fluorescent protein was found far away from the site of injection in the head and the tail sections. Thus, electroporation in adult zebrafish provides a rapid way of testing the behavior of gene sequences in the whole organism.

  4. Phase IIb trial of in vivo electroporation mediated dual-plasmid hepatitis B virus DNA vaccine in chronic hepatitis B patients under lamivudine therapy

    Science.gov (United States)

    Yang, Fu-Qiang; Rao, Gui-Rong; Wang, Gui-Qiang; Li, Yue-Qi; Xie, Yao; Zhang, Zhan-Qing; Deng, Cun-Liang; Mao, Qing; Li, Jun; Zhao, Wei; Wang, Mao-Rong; Han, Tao; Chen, Shi-Jun; Pan, Chen; Tan, De-Ming; Shang, Jia; Zhang, Ming-Xiang; Zhang, Yue-Xin; Yang, Ji-Ming; Chen, Guang-Ming

    2017-01-01

    AIM To assess the efficacy and safety of in vivo electroporation (EP)-mediated dual-plasmid hepatitis B virus (HBV) DNA vaccine vs placebo for sequential combination therapy with lamivudine (LAM) in patients with chronic hepatitis B. METHODS Two hundred and twenty-five patients were randomized to receive either LAM + vaccine (vaccine group, n = 109) or LAM + placebo (control group, n = 116). LAM treatment lasted 72 wk. Patients received the DNA vaccine or placebo by intramuscular injection mediated by EP at weeks 12 (start of treatment with vaccine or placebo, SOT), 16, 24, and 36 (end of treatment with vaccine or placebo, EOT). RESULTS In the modified intent-to-treat population, more patients had a decrease in HBV DNA > 2 log10 IU/mL in the vaccine group at week 12 after EOT compared with the control group. A trend toward a difference in the number of patients with undetectable HBV DNA at week 28 after EOT was obtained. Adverse events were similar. In the dynamic per-protocol set, which excluded adefovir (ADV) add-on cases at each time point instantly after ADV administration due to LAM antiviral failure, more patients had a decrease in HBV DNA > 2 log10 IU/mL in the vaccine group at week 12 and 28 after EOT compared with the control group. More patients with undetectable HBV DNA at week 28 after EOT in the vaccine group were also observed. Among patients with a viral load < 1000 copies/mL at week 12, more patients achieved HBeAg seroconversion in the vaccine group than among controls at week 36 after EOT, as well as less virological breakthrough and YMDD mutations. CONCLUSION The primary endpoint was not achieved using the HBV DNA vaccine. The HBV DNA vaccine could only be beneficial in subjects that have achieved initial virological response under LAM chemotherapy. PMID:28127204

  5. In vivo suppression of vein graft disease by nonviral, electroporation-mediated, gene transfer of tissue inhibitor of metalloproteinase-1 linked to the amino terminal fragment of urokinase (TIMP-1.ATF), a cell-surface directed matrix metalloproteinase inhibitor.

    Science.gov (United States)

    Eefting, Daniel; de Vries, Margreet R; Grimbergen, Jos M; Karper, Jacco C; van Bockel, J Hajo; Quax, Paul H A

    2010-02-01

    Smooth muscle cell (SMC) migration and proliferation are important in the development of intimal hyperplasia, the major cause of vein graft failure. Proteases of the plasminogen activator (PA) system and of the matrix metalloproteinase (MMP) system are pivotal in extracellular matrix degradation and, by that, SMC migration. Previously, we demonstrated that inhibition of both protease systems simultaneously with viral gene delivery of the hybrid protein TIMP-1.ATF, consisting of the tissue inhibitor of metalloproteinase-1 (TIMP-1) and the receptor-binding amino terminal fragment (ATF) of urokinase, reduces SMC migration and neointima formation in an in vitro restenosis model using human saphenous vein cultures more efficiently than both protease systems separately. Because use of viral gene delivery is difficult in clinical application, this study used nonviral delivery of TIMP-1.ATF plasmid to reduce vein graft disease in a murine bypass model. Nonviral gene transfer by electroporation was used to avert major disadvantages of viral gene delivery, such as immune responses and short-term expression. Plasmids encoding ATF, TIMP-1, TIMP-1.ATF, or luciferase, as a control, were injected and electroporated in both calf muscles of hypercholesterolemic apolipoprotein E3-Leiden (APOE*3Leiden) mice (n = 8). One day after electroporation, a venous interposition of a donor mouse was placed into the carotid artery of a recipient mouse. In this model, vein graft thickening develops with features of accelerated atherosclerosis. Vein grafts were harvested 4 weeks after electroporation and surgery, and histologic analysis of the vessel wall was performed. Electroporation-mediated overexpression of the plasmid vectors resulted in a prolonged expression of the transgenes and resulted in a significant reduction of vein graft thickening (ATF: 36% +/- 9%, TIMP-1: 49% +/- 5%, TIMP-1.ATF: 58% +/- 5%; P ATF-treated mice. Intramuscular electroporation of TIMP-1.ATF inhibits vein graft

  6. Perspectives on Transdermal Electroporation

    Science.gov (United States)

    Ita, Kevin

    2016-01-01

    Transdermal drug delivery offers several advantages, including avoidance of erratic absorption, absence of gastric irritation, painlessness, noninvasiveness, as well as improvement in patient compliance. With this mode of drug administration, there is no pre-systemic metabolism and it is possible to increase drug bioavailability and half-life. However, only a few molecules can be delivered across the skin in therapeutic quantities. This is because of the hindrance provided by the stratum corneum. Several techniques have been developed and used over the last few decades for transdermal drug delivery enhancement. These include sonophoresis, iontophoresis, microneedles, and electroporation. Electroporation, which refers to the temporary perturbation of the skin following the application of high voltage electric pulses, has been used to increase transcutaneous flux values by several research groups. In this review, transdermal electroporation is discussed and the use of the technique for percutaneous transport of low and high molecular weight compounds described. This review also examines our current knowledge regarding the mechanisms of electroporation and safety concerns arising from the use of this transdermal drug delivery technique. Safety considerations are especially important because electroporation utilizes high voltage pulses which may have deleterious effects in some cases. PMID:26999191

  7. Perspectives on Transdermal Electroporation

    Directory of Open Access Journals (Sweden)

    Kevin Ita

    2016-03-01

    Full Text Available Transdermal drug delivery offers several advantages, including avoidance of erratic absorption, absence of gastric irritation, painlessness, noninvasiveness, as well as improvement in patient compliance. With this mode of drug administration, there is no pre-systemic metabolism and it is possible to increase drug bioavailability and half-life. However, only a few molecules can be delivered across the skin in therapeutic quantities. This is because of the hindrance provided by the stratum corneum. Several techniques have been developed and used over the last few decades for transdermal drug delivery enhancement. These include sonophoresis, iontophoresis, microneedles, and electroporation. Electroporation, which refers to the temporary perturbation of the skin following the application of high voltage electric pulses, has been used to increase transcutaneous flux values by several research groups. In this review, transdermal electroporation is discussed and the use of the technique for percutaneous transport of low and high molecular weight compounds described. This review also examines our current knowledge regarding the mechanisms of electroporation and safety concerns arising from the use of this transdermal drug delivery technique. Safety considerations are especially important because electroporation utilizes high voltage pulses which may have deleterious effects in some cases.

  8. Preparing clinical-grade myeloid dendritic cells by electroporation-mediated transfection of in vitro amplified tumor-derived mRNA and safety testing in stage IV malignant melanoma

    Directory of Open Access Journals (Sweden)

    Allred Jacob B

    2006-08-01

    Full Text Available Abstract Background Dendritic cells (DCs have been used as vaccines in clinical trials of immunotherapy of cancer and other diseases. Nonetheless, progress towards the use of DCs in the clinic has been slow due in part to the absence of standard methods for DC preparation and exposure to disease-associated antigens. Because different ex vivo exposure methods can affect DC phenotype and function differently, we studied whether electroporation-mediated transfection (electrotransfection of myeloid DCs with in vitro expanded RNA isolated from tumor tissue might be feasible as a standard physical method in the preparation of clinical-grade DC vaccines. Methods We prepared immature DCs (IDCs from CD14+ cells isolated from leukapheresis products and extracted total RNA from freshly resected melanoma tissue. We reversely transcribed the RNA while attaching a T7 promoter to the products that we subsequently amplified by PCR. We transcribed the amplified cDNA in vitro and introduced the expanded RNA into IDCs by electroporation followed by DC maturation and cryopreservation. Isolated and expanded mRNA was analyzed for the presence of melanoma-associated tumor antigens gp100, tyrosinase or MART1. To test product safety, we injected five million DCs subcutaneously at three-week intervals for up to four injections into six patients suffering from stage IV malignant melanoma. Results Three preparations contained all three transcripts, one isolate contained tyrosinase and gp100 and one contained none. Electrotransfection of DCs did not affect viability and phenotype of fresh mature DCs. However, post-thaw viability was lower (69 ± 12 percent in comparison to non-electroporated cells (82 ± 12 percent; p = 0.001. No patient exhibited grade 3 or 4 toxicity upon DC injections. Conclusion Standardized preparation of viable clinical-grade DCs transfected with tumor-derived and in vitro amplified mRNA is feasible and their administration is safe.

  9. CHO-S Antibody Titers >1 Gram/Liter Using Flow Electroporation-Mediated Transient Gene Expression followed by Rapid Migration to High-Yield Stable Cell Lines

    OpenAIRE

    Steger, Krista; Brady, James; WANG, WEILI; Duskin, Meg; Donato, Karen; Peshwa, Madhusudan

    2015-01-01

    In recent years, researchers have turned to transient gene expression (TGE) as an alternative to CHO stable cell line generation for early-stage antibody development. Despite advances in transfection methods and culture optimization, the majority of CHO-based TGE systems produce insufficient antibody titers for extensive use within biotherapeutic development pipelines. Flow electroporation using the MaxCyte STX Scalable Transfection System is a highly efficient, scalable means of CHO-based TG...

  10. CHO-S antibody titers >1 gram/liter using flow electroporation-mediated transient gene expression followed by rapid migration to high-yield stable cell lines.

    Science.gov (United States)

    Steger, Krista; Brady, James; Wang, Weili; Duskin, Meg; Donato, Karen; Peshwa, Madhusudan

    2015-04-01

    In recent years, researchers have turned to transient gene expression (TGE) as an alternative to CHO stable cell line generation for early-stage antibody development. Despite advances in transfection methods and culture optimization, the majority of CHO-based TGE systems produce insufficient antibody titers for extensive use within biotherapeutic development pipelines. Flow electroporation using the MaxCyte STX Scalable Transfection System is a highly efficient, scalable means of CHO-based TGE for gram-level production of antibodies without the need for specialized expression vectors or genetically engineered CHO cell lines. CHO cell flow electroporation is easily scaled from milligram to multigram quantities without protocol reoptimization while maintaining transfection performance and antibody productivity. In this article, data are presented that demonstrate the reproducibility, scalability, and antibody production capabilities of CHO-based TGE using the MaxCyte STX. Data show optimization of posttransfection parameters such as cell density, media composition, and feed strategy that result in secreted antibody titers >1 g/L and production of multiple grams of antibody within 2 weeks of a single CHO-S cell transfection. In addition, data are presented to demonstrate the application of scalable electroporation for the rapid generation of high-yield stable CHO cell lines to bridge the gap between early- and late-stage antibody development activities.

  11. 探讨电穿孔法介导转染32DP210细胞的参数%Investigation on optimal parameters of electroporation-mediated transfection in 32DP210

    Institute of Scientific and Technical Information of China (English)

    吕远栋; 王雅珍; 徐伟红; 陶萍华; 林兵

    2012-01-01

    Objective:To find the best parameters to transfect plasmid Pg into 32DP210 cells. Methods; Iipo-fectamineTM2000, transfast, liposome DMRIC — E transfection and electroporation were compared for transfecting plasmid pG,a plasmid that expressed G418 resistance protein and green fluorescent protein (GFP) , into 32DP210 cells. The expression of GFP was observed and the transient transfection efficiency was measured after the transfection by fluorescence microscope. The stable transfected cells were screened by G418. Results: The optimum conditions for laboratory electroporationmediated 32DP210 cell transfection were; power transfer voltage 270 V, capacitance of 950 μF, DNA dose of 14 μg. Transfection efficiency of electroporation after 48 h was 50. 81% , while the other three methods were less than 3%. Conclusion; Electroporation is the best way to stably transfect plasmid pG into 32DP210 cells., c%目的:采用电穿孔法将质粒pGenesil -1(简称pG)转染32DP210细胞,从中寻找最佳的参数.方法:分别采用LipofectamineTM2000、transfast、脂质体DMRIC -E和电穿孔法,将能表达G418抗性蛋白和绿色荧光蛋白(GFP)的质粒pG转染32DP210细胞;荧光显微镜下观察GFP表达并测定瞬时转染率;通过G418筛选得到稳定转染的细胞.结果:本实验室电穿孔法介导的32DP210细胞转染其最佳电转条件为电压270 V,电容950μF,DNA剂量14 μg.48 h后电穿孔法的转染效率达50.81%,其它三种方法转染效率都小于3%.结论:用电穿孔法将质粒pG转染32DP210细胞是最佳的转染方法.

  12. Handbook of electroporation

    CERN Document Server

    2017-01-01

    This major reference work is a one-shot knowledge base on electroporation and the use of pulsed electric fields of high intensity and their use in biology, medicine, biotechnology, and food and environmental technologies. The Handbook offers a widespread and well-structured compilation of 156 chapters ranging from the foundations to applications in industry and hospital. It is edited and written by most prominent researchers in the field. With regular updates and growing in its volume it is suitable for academic readers and researchers regardless of their disciplinary expertise, and will also be accessible to students and serious general readers. The Handbook's 276 authors have established scholarly credentials and come from a wide range of disciplines. This is crucially important in a highly interdisciplinary field of electroporation and the use of pulsed electric fields of high intensity and its applications in different fields from medicine, biology, food proce ssing, agriculture, process engineering, en...

  13. High-frequency submicrosecond electroporator

    Directory of Open Access Journals (Sweden)

    Vitalij Novickij

    2016-05-01

    Full Text Available In this work, we present a novel electroporator which is capable of generating single and bursts of high power (3 kV, 60 A square wave pulses of variable duration (100 ns to 1 ms with predefined repetition frequency (1 Hz to 3.5 MHz. The proposed synchronized crowbar implementation ensures a constant pulse rise and fall times, which are independent from the load, thus highly relevant in electroporation. The electroporator was successfully tested for the inactivation of the human pathogen Candida albicans. The device is compatible with standard commercial electroporation cuvettes.

  14. Inhibitory effect of Ca2+ on in vivo gene transfer by electroporation

    Institute of Scientific and Technical Information of China (English)

    Yong-gang ZHAO; Hui-li LU; Jin-liang PENG; Yu-hong XU

    2006-01-01

    Aim:To investigate the specific effects of Ca2+ on transgene expression during electroporation-mediated gene transfer in mice.Methods:Skeletal muscle and skin were subjected to in vivo electroporation with a luciferase reporter plasmid,with or Without Ca2+ and various other ions.Resuits:For in vivo electroporation,the presence of just 10 mmol/L Ca2+ in the DNA solution drastically reduced the resulting transgene expression,to less than 5% of control values.Only Ca2+,not other ions,caused inhibition,and the effect was not tissue specific.More surprisingly.even when Ca2+ ions were delivered by electroporation before or after DNA administration,similar effects were still observed.Conelusion:The inhibitory effect of Ca2+ on in vivo gene transfer by electroporation is specific,ie,the inhibitory effect may be related to the cell membrane properties after electroporation and the subsequent resealing event.

  15. Electroporation-induced electrosensitization.

    Directory of Open Access Journals (Sweden)

    Olga N Pakhomova

    Full Text Available BACKGROUND: Electroporation is a method of disrupting the integrity of cell membrane by electric pulses (EPs. Electrical modeling is widely employed to explain and study electroporation, but even most advanced models show limited predictive power. No studies have accounted for the biological consequences of electroporation as a factor that alters the cell's susceptibility to forthcoming EPs. METHODOLOGY/PRINCIPAL FINDINGS: We focused first on the role of EP rate for membrane permeabilization and lethal effects in mammalian cells. The rate was varied from 0.001 to 2,000 Hz while keeping other parameters constant (2 to 3,750 pulses of 60-ns to 9-µs duration, 1.8 to 13.3 kV/cm. The efficiency of all EP treatments was minimal at high rates and started to increase gradually when the rate decreased below a certain value. Although this value ranged widely (0.1-500 Hz, it always corresponded to the overall treatment duration near 10 s. We further found that longer exposures were more efficient irrespective of the EP rate, and that splitting a high-rate EP train in two fractions with 1-5 min delay enhanced the effects severalfold. CONCLUSIONS/SIGNIFICANCE: For varied experimental conditions, EPs triggered a delayed and gradual sensitization to EPs. When a portion of a multi-pulse exposure was delivered to already sensitized cells, the overall effect markedly increased. Because of the sensitization, the lethality in EP-treated cells could be increased from 0 to 90% simply by increasing the exposure duration, or the exposure dose could be reduced twofold without reducing the effect. Many applications of electroporation can benefit from accounting for sensitization, by organizing the exposure either to maximize sensitization (e.g., for sterilization or, for other applications, to completely or partially avoid it. In particular, harmful side effects of electroporation-based therapies (electrochemotherapy, gene therapies, tumor ablation include convulsions

  16. Electroporation in veterinary oncology.

    Science.gov (United States)

    Impellizeri, J; Aurisicchio, L; Forde, P; Soden, D M

    2016-11-01

    Cancer treatments in veterinary medicine continue to evolve beyond the established standard therapies of surgery, chemotherapy and radiation therapy. New technologies in cancer therapy include a targeted mechanism to open the cell membrane based on electroporation, driving therapeutic agents, such as chemotherapy (electro-chemotherapy), for local control of cancer, or delivery of gene-based products (electro-gene therapy), directly into the cancer cell to achieve systemic control. This review examines electrochemotherapy and electro-gene therapy in veterinary medicine and considers future directions and applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Electroporation System for Sterilizing Water

    Science.gov (United States)

    Schlager, Kenneth J.

    2005-01-01

    A prototype of an electroporation system for sterilizing wastewater or drinking water has been developed. In electroporation, applied electric fields cause transient and/or permanent changes in the porosities of living cells. Electroporation at lower field strengths can be exploited to increase the efficiency of chemical disinfection (as in chlorination). Electroporation at higher field strengths is capable of inactivating and even killing bacteria and other pathogens, without use of chemicals. Hence, electroporation is at least a partial alternative to chlorination. The transient changes that occur in micro-organisms at lower electric-field strengths include significantly increased uptake of ions and molecules. Such increased uptake makes it possible to achieve disinfection at lower doses of chemicals (e.g., chlorine or ozone) than would otherwise be needed. Lower doses translate to lower costs and reduced concentrations of such carcinogenic chemical byproducts as trichloromethane. Higher electric fields cause cell membranes to lose semipermeability and thereby become unable to function as selective osmotic barriers between the cells and the environment. This loss of function is the cause of the cell death at higher electric-field intensities. Experimental evidence does not indicate cell lysis but, rather, combined leaking of cell proteins out of the cells as well as invasion of foreign chemical compounds into the cells. The concept of electroporation is not new: it has been applied in molecular biology and genetic engineering for decades. However, the laboratory-scale electroporators used heretofore have been built around small (400-microliter) cuvettes, partly because the smallness facilitates the generation of electric fields of sufficient magnitude to cause electroporation. Moreover, most laboratory- scale electroporators have been designed for testing static water. In contrast, the treatment cell in the present system is much larger and features a flow

  18. Electroporation of the Testis

    Science.gov (United States)

    Yomogida, Kentaro

    The mature mammalian testis is a marvelous organ that produces numerous sperm cells during its reproductive phase. This biologically significant process consists of three steps: stem cell self-renewal and differentiation, meiosis and genetic recombination, and haploid cell morphogenesis into sperm (Russell et al., 1990). The first step provides a good model for investigating the molecular mechanism of stem cell regulation. Currently, the mechanism underlying sperm cell production is a very exciting topic in regenerative medicine (Lensch et al. 2007; Okita et al., 2007). The spermatogonial stem cell system has several advantages, including the easy histological identification of stem cells (Russell et al., 1990), a clear relationship between stem cells and the supporting Sertoli cells, which provide a stem cell niche (Tadokoro et al., 2002; Yomogida et al., 2003), and a transplantation assay for stem cell activity (Oatley & Brinster, 2006). Although germline stem (GS) cells derived from the gonocytes in newborn testis constitute a suitable in vitro system for investigating the properties of spermatogonial stem cells (Kanatsu-Shinohara et al., 2003, 2004), studies using living mammalian testes continue to provide information regarding the roles of the stem cell niche. In vivo electroporation of the supporting cells in the testis will expand our ability to study it.

  19. Electrical breakdown in tissue electroporation.

    Science.gov (United States)

    Guenther, Enric; Klein, Nina; Mikus, Paul; Stehling, Michael K; Rubinsky, Boris

    2015-11-27

    Electroporation, the permeabilization of the cell membrane by brief, high electric fields, has become an important technology in medicine for diverse application ranging from gene transfection to tissue ablation. There is ample anecdotal evidence that the clinical application of electroporation is often associated with loud sounds and extremely high currents that exceed the devices design limit after which the devices cease to function. The goal of this paper is to elucidate and quantify the biophysical and biochemical basis for this phenomenon. Using an experimental design that includes clinical data, a tissue phantom, sound, optical, ultrasound and MRI measurements, we show that the phenomenon is caused by electrical breakdown across ionized electrolysis produced gases near the electrodes. The breakdown occurs primarily near the cathode. Electrical breakdown during electroporation is a biophysical phenomenon of substantial importance to the outcome of clinical applications. It was ignored, until now.

  20. Microfluidic electroporation for cellular analysis and delivery.

    Science.gov (United States)

    Geng, Tao; Lu, Chang

    2013-10-01

    Electroporation is a simple yet powerful technique for breaching the cell membrane barrier. The applications of electroporation can be generally divided into two categories: the release of intracellular proteins, nucleic acids and other metabolites for analysis and the delivery of exogenous reagents such as genes, drugs and nanoparticles with therapeutic purposes or for cellular manipulation. In this review, we go over the basic physics associated with cell electroporation and highlight recent technological advances on microfluidic platforms for conducting electroporation. Within the context of its working mechanism, we summarize the accumulated knowledge on how the parameters of electroporation affect its performance for various tasks. We discuss various strategies and designs for conducting electroporation at the microscale and then focus on analysis of intracellular contents and delivery of exogenous agents as two major applications of the technique. Finally, an outlook for future applications of microfluidic electroporation in increasingly diverse utilities is presented.

  1. Optimization of a plasmid electroporation protocol for Aeromonas salmonicida subsp. salmonicida.

    Science.gov (United States)

    Dallaire-Dufresne, Stéphanie; Emond-Rheault, Jean-Guillaume; Attéré, Sabrina A; Tanaka, Katherine H; Trudel, Mélanie V; Frenette, Michel; Charette, Steve J

    2014-03-01

    Aeromonas salmonicida subsp. salmonicida is a major fish pathogen. Molecular tools are required to study the virulence and genomic stability of this bacterium. An efficient electroporation-mediated transformation protocol for A. salmonicida subsp. salmonicida would make genetic studies faster and easier. In the present study, we designed the 4.1-kb pSDD1 plasmid as a tool for optimizing an electroporation protocol for A. salmonicida subsp. salmonicida. We systematically tested the electroporation conditions to develop a protocol that generates the maximum number of transformants. Under these optimal conditions (25 kV/cm, 200 Ω, 25 μF), we achieved an electroporation efficiency of up to 1×10(5) CFU/μg DNA. The electroporation protocol was also tested using another plasmid of 10.6-kb and three different strains of A. salmonicida subsp. salmonicida. The strains displayed significant differences in their electro-transformation competencies. Strain 01-B526 was the easiest to electroporate, especially with the pSDD1 plasmid. This plasmid was stably maintained in the 01-B526 transformants, as were the native plasmids, but could be easily cured by removing the selection conditions. This is the first efficient electroporation protocol reported for A. salmonicida subsp. salmonicida, and offers new possibilities for studying this bacterium.

  2. Single cell electroporation on chip

    NARCIS (Netherlands)

    Valero, Ana

    2006-01-01

    In this thesis the results of the development of microfluidic cell trap devices for single cell electroporation are described, which are to be used for gene transfection. The performance of two types of Lab-on-a-Chip trapping devices was tested using beads and cells, whereas the functionality for si

  3. Electrolytic Effects During Tissue Ablation by Electroporation.

    Science.gov (United States)

    Rubinsky, Liel; Guenther, Enric; Mikus, Paul; Stehling, Michael; Rubinsky, Boris

    2016-10-01

    Nonthermal irreversible electroporation is a new tissue ablation technique that consists of applying pulsed electric fields across cells to induce cell death by creating permanent defects in the cell membrane. Nonthermal irreversible electroporation is of interest because it allows treatment near sensitive tissue structures such as blood vessels and nerves. Two recent articles report that electrolytic reaction products at electrodes can be combined with electroporation pulses to augment and optimize tissue ablation. Those articles triggered a concern that the results of earlier studies on nonthermal irreversible electroporation may have been tainted by unaccounted for electrolytic effects. The goal of this study was to reexamine previous studies on nonthermal irreversible electroporation in the context of these articles. The study shows that the results from some of the earlier studies on nonthermal irreversible electroporation were affected by unaccounted for electrolysis, in particular the research with cells in cuvettes. It also shows that tissue ablation ascribed in the past to irreversible electroporation is actually caused by at least 3 different cytotoxic effects: irreversible electroporation without electrolysis, irreversible electroporation combined with electrolysis, and reversible electroporation combined with electrolysis. These different mechanisms may affect cell and tissue ablation in different ways, and the effects may depend on various clinical parameters such as the polarity of the electrodes, the charge delivered (voltage, number, and length of pulses), and the distance of the target tissue from the electrodes. Current clinical protocols employ ever-increasing numbers of electroporation pulses to values that are now an order of magnitude larger than those used in our first fundamental nonthermal irreversible electroporation studies in tissues. The different mechanisms of cell death, and the effect of the clinical parameters on the mechanisms may

  4. Tumor ablation with irreversible electroporation.

    Directory of Open Access Journals (Sweden)

    Bassim Al-Sakere

    Full Text Available We report the first successful use of irreversible electroporation for the minimally invasive treatment of aggressive cutaneous tumors implanted in mice. Irreversible electroporation is a newly developed non-thermal tissue ablation technique in which certain short duration electrical fields are used to permanently permeabilize the cell membrane, presumably through the formation of nanoscale defects in the cell membrane. Mathematical models of the electrical and thermal fields that develop during the application of the pulses were used to design an efficient treatment protocol with minimal heating of the tissue. Tumor regression was confirmed by histological studies which also revealed that it occurred as a direct result of irreversible cell membrane permeabilization. Parametric studies show that the successful outcome of the procedure is related to the applied electric field strength, the total pulse duration as well as the temporal mode of delivery of the pulses. Our best results were obtained using plate electrodes to deliver across the tumor 80 pulses of 100 micros at 0.3 Hz with an electrical field magnitude of 2500 V/cm. These conditions induced complete regression in 12 out of 13 treated tumors, (92%, in the absence of tissue heating. Irreversible electroporation is thus a new effective modality for non-thermal tumor ablation.

  5. Establishment of an in vivo electroporation method into postnatal newborn neurons in the dentate gyrus.

    Science.gov (United States)

    Ito, Hidenori; Morishita, Rika; Iwamoto, Ikuko; Nagata, Koh-ichi

    2014-12-01

    Electroporation-mediated gene transfer has been developed for the analysis of mammalian brain development in vivo. Indeed, in utero electroporation method is widely used for the investigation of the mouse embryonic cortical development while in vivo electroporation using neonatal mouse brain is employed for the analysis of the rostral migratory stream (RMS) and postnatal olfactory neurogenesis. In the present study, we established a stable gene-transfer method to dentate gyrus (DG) neurons by carefully determining the in vivo electroporation conditions, such as position and direction of electrode, voltage for electric pulses, and interval between electroporation and sample preparation. Consequently, GFP-positive cells in DG were observed to extend branched dendrites and long axons into the molecular layer and the hilus, respectively, 21 days after electrporation. They were morphologically identified as dentate granule neurons with many protrusions on dendrites, and some of them had wide head and thin neck that resembled matured mushroom spines. Expression of GFP in dentate neurons sustained for at least 9 months after electroporation under our experimental conditions. Taken together, the method developed here could be a powerful new tool for the analysis of the postnatal DG development.

  6. Single cell electroporation using microfluidic devices

    NARCIS (Netherlands)

    Le Gac, S.; Berg, van den A.; Lindstrom, S.; Andersson-Svahn, S.M.H.

    2012-01-01

    Electroporation is a powerful technique to increase the permeability of cell membranes and subsequently introduce foreign materials into cells. Pores are created in the cell membrane upon application of an electric fi eld (kV/cm). Most applications employ bulk electroporation, at the scale of 1 mL o

  7. Electroporation-based applications in biotechnology.

    Science.gov (United States)

    Kotnik, Tadej; Frey, Wolfgang; Sack, Martin; Haberl Meglič, Saša; Peterka, Matjaž; Miklavčič, Damijan

    2015-08-01

    Electroporation is already an established technique in several areas of medicine, but many of its biotechnological applications have only started to emerge; we review here some of the most promising. We outline electroporation as a phenomenon and then proceed to applications, first outlining the best established - the use of reversible electroporation for heritable genetic modification of microorganisms (electrotransformation), and then explore recent advances in applying electroporation for inactivation of microorganisms, extraction of biomolecules, and fast drying of biomass. Although these applications often aim to upscale to the industrial and/or clinical level, we also outline some important chip-scale applications of electroporation. We conclude our review with a discussion of the main challenges and future perspectives.

  8. Electroporation in food processing and biorefinery.

    Science.gov (United States)

    Mahnič-Kalamiza, Samo; Vorobiev, Eugène; Miklavčič, Damijan

    2014-12-01

    Electroporation is a method of treatment of plant tissue that due to its nonthermal nature enables preservation of the natural quality, colour and vitamin composition of food products. The range of processes where electroporation was shown to preserve quality, increase extract yield or optimize energy input into the process is overwhelming, though not exhausted; e.g. extraction of valuable compounds and juices, dehydration, cryopreservation, etc. Electroporation is--due to its antimicrobial action--a subject of research as one stage of the pasteurization or sterilization process, as well as a method of plant metabolism stimulation. This paper provides an overview of electroporation as applied to plant materials and electroporation applications in food processing, a quick summary of the basic technical aspects on the topic, and a brief discussion on perspectives for future research and development in the field. The paper is a review in the very broadest sense of the word, written with the purpose of orienting the interested newcomer to the field of electroporation applications in food technology towards the pertinent, highly relevant and more in-depth literature from the respective subdomains of electroporation research.

  9. Cell viability of bovine spermatozoa subjected to DNA electroporation and DNAse I treatment.

    Science.gov (United States)

    Cavalcanti, Paulo Varoni; Milazzotto, Marcella Pecora; Simões, Renata; Nichi, Marcilio; de Oliveira Barros, Flavia Regina; Visintin, Jose Antonio; Assumpção, Mayra Elena Ortiz D'Avila

    2016-04-15

    Many mechanisms involved in sperm-mediated gene transfer (SMGT) are still unknown. It is still a matter of debate whether exogenous DNA fragments incorporated by the embryo are originated from those bound to the sperm membrane or by those that penetrated the intracellular compartment. In an attempt to elucidate the transmission mechanism of exogenous DNA molecules by sperm, some authors suggested a treatment with DNAse I to remove DNA molecules outside the sperm. But little is known regarding the effects of DNAse I treatment on sperm viability and its impact on sperm organelles. An important aspect of the SMGT technique is the amount of exogenous DNA incubated with sperm, which may influence the internalization rate. Due to the inconsistencies found in literature, this work aimed to contribute to bovine sperm physiology knowledge evaluating the effects of different DNA concentrations, electroporation, and DNAse I treatments on sperm viability characteristics, DNA uptake, and IVF. For that, the effects of different concentrations of exogenous DNA (250, 500 and 1000 ng/10(6) cells) and incubation or electroporation were tested on sperm functional characteristics and in vitro embryo production. No effect of DNA concentration was observed on uptake, plasma membrane integrity, and mitochondrial membrane potential. The addition of exogenous DNA induced a decrease on acrosomal lesion in the 500-ng group when compared to the control. Cells incubated with DNA, electroporated, and treated with DNAse I presented a deleterious influence on mitochondrial membrane potential. In vitro fertilization was made with 1000 ng of DNA, sperm cells incubated or electroporated followed by DNAse I treatment. No significant difference was found in cleavage rate. Blastocyst rates were 24.36% for the control; 19.65% for incubated; 3.5% for electroporated control; and 17.40% for electroporated. There is a significant difference in blastocyst rate between the control and electroporated control

  10. Enhancing transdermal drug delivery with electroporation.

    Science.gov (United States)

    Wong, Tak-Wah; Ko, Shu-Fen; Hui, Sek-Wen

    2008-01-01

    The application of electroporation to enhance transdermal delivery has opened up a new possibility to introduce larger molecules such as peptide hormones and vaccines as well as minigenes and RNAi etc. through the transdermal route. Many devices have been developed to deliver the pulse electric field needed to permeate the skin. These devices include both non-puncturing surface electrodes as well as puncturing electrodes of different geometrical arrangements. The latter type uses electroporation only to increase uptake of molecules injected through the puncturing electrode or syringe. Different electroporation protocols have been developed to maximize transport, uptake and minimizing pain. Synergistic effect of chemical enhancers and physical (sonic, vibrational and thermal) treatments are used to enhance the transport. This article reviews the patents pertaining to the instrumentation as well as application protocols of transdermal delivery, uptake enhancement and interstitial fluid sampling by electroporation.

  11. High-Efficiency Transfection of Primary Human and Mouse T Lymphocytes Using RNA Electroporation

    Science.gov (United States)

    Zhao, Yangbing; Zheng, Zhili; Cohen, Cyrille J.; Gattinoni, Luca; Palmer, Douglas C.; Restifo, Nicholas P.; Rosenberg, Steven A.; Morgan, Richard A.

    2006-01-01

    The use of nonviral gene transfer methods in primary lymphocytes has been hampered by low gene transfer efficiency and high transfection-related toxicity. In this report, high gene transfection efficiency with low transfection-related toxicity was achieved by electroporation using in vitro-transcribed mRNA. Using these methods, >90% transgene expression with >80% viable cells was observed in stimulated primary human and murine T lymphocytes transfected with GFP or mCD62L. Electroporation of unstimulated human PBMCs or murine splenocytes with GFP RNA yielded 95 and 56% GFP+ cells, respectively. Electroporation of mRNA for NY-ESO-1, MART-1, and p53 antigen-specific TCRs into human T lymphocytes redirected these lymphocytes to recognize melanoma cell lines in an MHC-restricted manner. The onset of gene expression was rapid (within 30 min) and durable (up to 7 days postelectroporation) using both GFP and TCR-mediated recognition of target cells. There was no adverse effect observed on the T lymphocytes subjected to RNA electroporation evaluated by cell growth rate, annexin-V staining of apoptotic cells, BrdU incorporation, tumor antigen-specific recognition or antigen-specific TCR affinity. The results of this study indicate that mRNA electroporation provides a powerful tool to introduce genes into both human and murine primary T lymphocytes. PMID:16140584

  12. Microfluidic Screening of Electric Fields for Electroporation

    Science.gov (United States)

    Garcia, Paulo A.; Ge, Zhifei; Moran, Jeffrey L.; Buie, Cullen R.

    2016-02-01

    Electroporation is commonly used to deliver molecules such as drugs, proteins, and/or DNA into cells, but the mechanism remains poorly understood. In this work a rapid microfluidic assay was developed to determine the critical electric field threshold required for inducing bacterial electroporation. The microfluidic device was designed to have a bilaterally converging channel to amplify the electric field to magnitudes sufficient to induce electroporation. The bacterial cells are introduced into the channel in the presence of SYTOX®, which fluorescently labels cells with compromised membranes. Upon delivery of an electric pulse, the cells fluoresce due to transmembrane influx of SYTOX® after disruption of the cell membranes. We calculate the critical electric field by capturing the location within the channel of the increase in fluorescence intensity after electroporation. Bacterial strains with industrial and therapeutic relevance such as Escherichia coli BL21 (3.65 ± 0.09 kV/cm), Corynebacterium glutamicum (5.20 ± 0.20 kV/cm), and Mycobacterium smegmatis (5.56 ± 0.08 kV/cm) have been successfully characterized. Determining the critical electric field for electroporation facilitates the development of electroporation protocols that minimize Joule heating and maximize cell viability. This assay will ultimately enable the genetic transformation of bacteria and archaea considered intractable and difficult-to-transfect, while facilitating fundamental genetic studies on numerous diverse microbes.

  13. Maximizing exosome colloidal stability following electroporation.

    Science.gov (United States)

    Hood, Joshua L; Scott, Michael J; Wickline, Samuel A

    2014-03-01

    Development of exosome-based semisynthetic nanovesicles for diagnostic and therapeutic purposes requires novel approaches to load exosomes with cargo. Electroporation has previously been used to load exosomes with RNA. However, investigations into exosome colloidal stability following electroporation have not been considered. Herein, we report the development of a unique trehalose pulse media (TPM) that minimizes exosome aggregation following electroporation. Dynamic light scattering (DLS) and RNA absorbance were employed to determine the extent of exosome aggregation and electroextraction post electroporation in TPM compared to common PBS pulse media or sucrose pulse media (SPM). Use of TPM to disaggregate melanoma exosomes post electroporation was dependent on both exosome concentration and electric field strength. TPM maximized exosome dispersal post electroporation for both homogenous B16 melanoma and heterogeneous human serum-derived populations of exosomes. Moreover, TPM enabled heavy cargo loading of melanoma exosomes with 5nm superparamagnetic iron oxide nanoparticles (SPION5) while maintaining original exosome size and minimizing exosome aggregation as evidenced by transmission electron microscopy. Loading exosomes with SPION5 increased exosome density on sucrose gradients. This provides a simple, label-free means of enriching exogenously modified exosomes and introduces the potential for MRI-driven theranostic exosome investigations in vivo.

  14. DNA vaccination in skin enhanced by electroporation.

    Science.gov (United States)

    Broderick, Kate E; Khan, Amir S; Sardesai, Niranjan Y

    2014-01-01

    DNA vaccines are a next generation branch of vaccines which offer major benefits over their conventional counterparts. However, to be effective in large mammals and humans, an enhancing delivery technology is required. Electroporation is a physical technique which results in improved delivery of large molecules through the cell membrane. In the case of plasmid DNA, electroporation enhances both the uptake and expression of the delivered DNA. The skin is an attractive tissue for DNA vaccination in a clinical setting due to the accessibility of the target, the ease of monitoring, and most importantly the immunocompetent nature of the dermis. Electroporation in the skin has the benefit of being minimally invasive and generally well tolerated. Previous studies have determined that optimized electroporation parameters (such as electrical field intensity, pulse length, pulse width, and plasmid formulation) majorly impact the efficiency of DNA delivery to the skin. We provide an overview of DNA vaccination in skin and muscle. In addition, we detail a protocol for the successful intradermal electroporation of plasmid DNA to guinea pig skin, an excellent dermatological animal model. The work detailed here suggests that the technique is safe and effective and could be highly applicable to a clinical setting.

  15. Agrobacterium-mediated transformation in chickpea (Cicer arietinum L.) with an insecticidal protein gene: optimisation of different factors.

    Science.gov (United States)

    Indurker, Shivani; Misra, Hari S; Eapen, Susan

    2010-07-01

    Agrobacterium-mediated transformation in chickpea was developed using strain LBA4404 carrying nptII, uidA and cryIAc genes and transformants selected on Murashige and Skoog's basal medium supplemented with benzyladenine, kinetin and kanamycin. Integration of transgenes was demonstrated using polymerase chain reaction and Southern blot hybridization of T0 plants. The expression of CryIAc delta endotoxin and GUS enzyme was shown by enzyme linked immunosorbent assay and histochemical assay respectively. The transgenic plants (T0) showed more tolerance to infection by Helicoverpa armigera compared to control plants. Various factors such as explant source, cultivar type, different preculture treatment period of explants, co-cultivation period, acetosyringone supplementation, Agrobacterium harboring different plasmids, vacuum infiltration and sonication treatment were tested to study the influence on transformation frequency. The results indicated that use of epicotyl as explant, cultivar ICCC37, Agrobacterium harboring plasmid pHS102 as vector, preculture of explant for 48 h, co-cultivation period of 2 days at 25°C and vacuum infiltration for 15 min produced the best transformation results. Sonication treatment of explants with Agrobacteria for 80 s was found to increase the frequency of transformation.

  16. Transformation of plant young proembryos by electroporation

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    It is first reported that plant young proembryos expressed exogenous reporter genes by electroporation. Young proembryos with 8-32 cells and globular proembryos with 250-400 cells could be isolated by enzymatic maceration combined with microdissection. After electroporation with GUS or GFP genes, the proembryos were cultured for 1-2 d in KM8p medium. At the field strength of electroporation 500-1 500 V/cm, blue reaction of GUS or green fluorescence of GFP could be observed in the proembryos. The highest transient expression frequency of young proembryos (2.2%) was obtained at the field strength of 750 V/cm, whereas the highest frequency of globular proembryos (5.9%) was obtained at the field strength of 1 250 V/cm. Taking the proportion of transformed cells in the whole cells of proembryos as efficient transformation frequency, the efficient transformation frequency of the young proembryos was 7 times that of the globular proembryos.

  17. Electroporation of cells in microfluidic droplets.

    Science.gov (United States)

    Zhan, Yihong; Wang, Jun; Bao, Ning; Lu, Chang

    2009-03-01

    Droplet-based microfluidics has raised a lot of interest recently due to its wide applications to screening biological/chemical assays with high throughput. Despite the advances on droplet-based assays involving cells, gene delivery methods that are compatible with the droplet platform have been lacking. In this report, we demonstrate a simple microfluidic device that encapsulates cells into aqueous droplets and then electroporates the encapsulated cells. The electroporation occurs when the cell-containing droplets (in oil) flow through a pair of microelectrodes with a constant voltage established in between. We investigate the parameters and characteristics of the electroporation. We demonstrate delivering enhanced green fluorescent protein (EGFP) plasmid into Chinese hamster ovary (CHO) cells. We envision the application of this technique to high-throughput functional genomics studies based on droplet microfluidics.

  18. Genetic transformation of plants by protoplast electroporation.

    Science.gov (United States)

    Bates, G W

    1994-10-01

    This article describes an optimized protocol for the electroporation of tobacco mesophyll protoplasts together with notes and data on the effects of various parameters and suggestions for work with protoplasts of other species. In this protocol, electroporation is achieved by means of electrical pulses from a high-voltage, capacitive-discharge unit. Procedures are described for measurement of protoplast viability with Evan's blue, the detection of transient expression of CAT and GUS gene plasmid constructs, and for the recovery of stable transformants based on selection for kanamycin resistance.

  19. Electroporation-based technologies for medicine: principles, applications, and challenges.

    Science.gov (United States)

    Yarmush, Martin L; Golberg, Alexander; Serša, Gregor; Kotnik, Tadej; Miklavčič, Damijan

    2014-07-11

    When high-amplitude, short-duration pulsed electric fields are applied to cells and tissues, the permeability of the cell membranes and tissue is increased. This increase in permeability is currently explained by the temporary appearance of aqueous pores within the cell membrane, a phenomenon termed electroporation. During the past four decades, advances in fundamental and experimental electroporation research have allowed for the translation of electroporation-based technologies to the clinic. In this review, we describe the theory and current applications of electroporation in medicine and then discuss current challenges in electroporation research and barriers to a more extensive spread of these clinical applications.

  20. Transformation of group A streptococci by electroporation

    NARCIS (Netherlands)

    Suvorov, Alexander; Kok, Jan; Venema, Gerhardus

    1988-01-01

    The introduction, via electroporation, of free plasmid DNA into three strains of Streptococcus pyogenes is described. The method is very simple and rapid and efficiencies vary from 1 × 10^3 to 4 × 10^4 per µg of DNA. The method was also used to introduce an integrative plasmid and transformants were

  1. Modeling of microvascular permeability changes after electroporation.

    Directory of Open Access Journals (Sweden)

    Selma Corovic

    Full Text Available Vascular endothelium selectively controls the transport of plasma contents across the blood vessel wall. The principal objective of our preliminary study was to quantify the electroporation-induced increase in permeability of blood vessel wall for macromolecules, which do not normally extravasate from blood into skin interstitium in homeostatic conditions. Our study combines mathematical modeling (by employing pharmacokinetic and finite element modeling approach with in vivo measurements (by intravital fluorescence microscopy. Extravasation of fluorescently labeled dextran molecules of two different sizes (70 kDa and 2000 kDa following the application of electroporation pulses was investigated in order to simulate extravasation of therapeutic macromolecules with molecular weights comparable to molecular weight of particles such as antibodies and plasmid DNA. The increase in blood vessel permeability due to electroporation and corresponding transvascular transport was quantified by calculating the apparent diffusion coefficients for skin microvessel wall (D [μm2/s] for both molecular sizes. The calculated apparent diffusion coefficients were D = 0.0086 μm2/s and D = 0.0045 μm2/s for 70 kDa and 2000 kDa dextran molecules, respectively. The results of our preliminary study have important implications in development of realistic mathematical models for prediction of extravasation and delivery of large therapeutic molecules to target tissues by means of electroporation.

  2. Electroporation-based DNA delivery technology

    DEFF Research Database (Denmark)

    Gothelf, A; Gehl, Julie

    2014-01-01

    DNA delivery to for example skin and muscle can easily be performed with electroporation. The method is efficient, feasible, and inexpensive and the future possibilities are numerous. Here we present our protocol for gene transfection to mouse skin using naked plasmid DNA and electric pulses....

  3. Modeling of microvascular permeability changes after electroporation.

    Science.gov (United States)

    Corovic, Selma; Markelc, Bostjan; Dolinar, Mitja; Cemazar, Maja; Jarm, Tomaz

    2015-01-01

    Vascular endothelium selectively controls the transport of plasma contents across the blood vessel wall. The principal objective of our preliminary study was to quantify the electroporation-induced increase in permeability of blood vessel wall for macromolecules, which do not normally extravasate from blood into skin interstitium in homeostatic conditions. Our study combines mathematical modeling (by employing pharmacokinetic and finite element modeling approach) with in vivo measurements (by intravital fluorescence microscopy). Extravasation of fluorescently labeled dextran molecules of two different sizes (70 kDa and 2000 kDa) following the application of electroporation pulses was investigated in order to simulate extravasation of therapeutic macromolecules with molecular weights comparable to molecular weight of particles such as antibodies and plasmid DNA. The increase in blood vessel permeability due to electroporation and corresponding transvascular transport was quantified by calculating the apparent diffusion coefficients for skin microvessel wall (D [μm2/s]) for both molecular sizes. The calculated apparent diffusion coefficients were D = 0.0086 μm2/s and D = 0.0045 μm2/s for 70 kDa and 2000 kDa dextran molecules, respectively. The results of our preliminary study have important implications in development of realistic mathematical models for prediction of extravasation and delivery of large therapeutic molecules to target tissues by means of electroporation.

  4. An Efficient Electroporation Protocol for the Genetic Modification of Mammalian Cells

    Science.gov (United States)

    Chicaybam, Leonardo; Barcelos, Camila; Peixoto, Barbara; Carneiro, Mayra; Limia, Cintia Gomez; Redondo, Patrícia; Lira, Carla; Paraguassú-Braga, Flávio; Vasconcelos, Zilton Farias Meira De; Barros, Luciana; Bonamino, Martin Hernán

    2017-01-01

    Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Electroporation using square-wave generating devices, like Lonza’s Nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work, we show that our in-house developed buffers, termed Chicabuffers, can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device, we electroporated 14 different cell lines and also primary cells, like mesenchymal stem cells and cord blood CD34+, providing optimized protocols for each of them. Moreover, when combined with sleeping beauty-based transposon system, long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells, facilitating the widespread adoption of this technology. PMID:28168187

  5. 电穿孔介导的pIRES-hVEGF165-EGFP转染对牵引成骨过程中早期血管生成的影响%Effect of electroporation mediated transfecting recombinant plasmid pIRES-hVEGF165-EGFP on angiogene-sis of distraction area during early mandibular distraction osteogenesis

    Institute of Scientific and Technical Information of China (English)

    吴国平; 黎德平; 何小川; 李盛华; 杨智慧; 廖毅; 郭力

    2010-01-01

    目的 探讨电穿孔介导的基因治疗对下领骨牵引成骨过程中早期血管生成的影响.方法 32只新西兰大白兔随机分为4组:质粒+电穿孔组(A组),质粒组(B组),生理盐水+电穿孔组(C组),空白对照组(D组).各组动物分别于注射后1、3、7、14 d处死,取牵引区组织进行组织学检查、电镜观察、CD34免疫组织化学染色及微血管密度检测.结果 A、B组血管内皮细胞呈增殖活跃状态;C、D组多数血管内皮细胞部分呈现退变及凋亡早期改变.免疫组化染色发现,转染后第1天血管壁内皮细胞浆CD34表达较弱;第3、7、14天,牵引区肉芽组织血管内皮细胞均出现CD34阳性表达.A组CD34阳性表达较B组强,A、B组的CD34表达持续阳性且呈上升趋势;C、D组表达最弱,CD34阳性表达维持在第1天水平上平稳波动.结论 电穿孔介导的pIRES-hVEGF165-EGFP重组质粒体内转染能够促进牵引区早期微血管的生成,使局部血管增生、渗入,增加骨断端的血流量.对调节和促进骨的生长和修复过程具有重要作用.%Objective To explore the effect of electroporation mediated gene therapy on angiogene-sis of the distraction area during early mandibular distraction osteogenesis (DO). Methods Thirty-two New-Zeland rabbits were randomly divided into 4 groups: group A: recombinant plasmid pIRES-VEGF165-EGFP and electroporation; group B: recombinant plasmid pIRES-VEGF165-EGFP; group C: normal saline (NS) and electroporation and group D: control group. The rabbits were sacrificed at 1d, 3d, 7d and 14d after injection, respectively. The distraction area tissue was removed for histological examination and electron microscopy, and immunohistochemical stain for CD34 was performed to detect the microvessel density. Results Generation of vascular endothelial cells (VEC) in the group A and group B were active, and majority of VEC in groups C and D took on early change of cataplasia and apoptosis. The

  6. Optimised polychromatic field-mediated suppression of H-atom tunnelling in a coupled symmetric double well: two-dimensional malonaldehyde model

    Science.gov (United States)

    Ghosh, Subhasree; Talukder, Srijeeta; Sen, Shrabani; Chaudhury, Pinaki

    2015-12-01

    The cis-cis isomerisation motion of malonaldehyde can be modelled as a symmetric double well coupled with an asymmetric double well, which includes the effect of the cis-trans out-of-plane motion on the cis-cis motion. We have presented an effective method for having control over the tunnelling dynamics of the symmetric double well which is coupled with the asymmetric double well by monitoring tunnelling splitting. When a suitable external field is allowed to interact with the system, the tunnelling splitting gets modified. As the external time perturbation is periodic in nature, the Floquet theory can be applied to calculate the quasi-energies of the perturbed system and hence the tunnelling splitting. The Floquet analysis is coupled with a stochastic optimiser in order to minimise the tunnelling splitting, which is related to slowering of the tunnelling process. The minimisation has been done by one of the stochastic optimisers, simulated annealing. Optimisation has been performed on the parameters which define the external polychromatic field, such as intensities and frequencies of the components of the polychromatic field. With the optimised sets of parameters, we have followed the dynamics of the system and have found suppression of tunnelling which is manifested by a much higher tunnelling time.

  7. DNA electroporation of multi-agent vaccines conferring protection against select agent challenge: TriGrid delivery system.

    Science.gov (United States)

    Keane-Myers, Andrea M; Bell, Matt; Hannaman, Drew; Albrecht, Mark

    2014-01-01

    Effective multi-agent/multivalent vaccines that confer protection against more than one disease are highly desirable to the patient and to health-care professionals. Electroporation of DNA vaccines, whereby tissues injected with DNA are subjected to localized electrical currents, is an ideal platform technology that achieves protective immune responses to multivalent vaccination. Here, we describe an electroporation-based immunization technique capable of administering a cocktail of DNA vaccinations in vivo. Immune response measurements, including protection from pathogen challenge and induction of antigen-specific antibody responses and cell-mediated immune responses, are also discussed.

  8. Non-Invasive Delivery of dsRNA into De-Waxed Tick Eggs by Electroporation

    Science.gov (United States)

    Ruiz, Newton; de Abreu, Leonardo Araujo; Parizi, Luís Fernando; Kim, Tae Kwon; Mulenga, Albert; Braz, Gloria Regina Cardoso; Vaz, Itabajara da Silva; Logullo, Carlos

    2015-01-01

    RNA interference-mediated gene silencing was shown to be an efficient tool for validation of targets that may become anti-tick vaccine components. Here, we demonstrate the application of this approach in the validation of components of molecular signaling cascades, such as the Protein Kinase B (AKT) / Glycogen Synthase Kinase (GSK) axis during tick embryogenesis. It was shown that heptane and hypochlorite treatment of tick eggs can remove wax, affecting corium integrity and but not embryo development. Evidence of AKT and GSK dsRNA delivery into de-waxed eggs of via electroporation is provided. Primers designed to amplify part of the dsRNA delivered into the electroporated eggs dsRNA confirmed its entry in eggs. In addition, it was shown that electroporation is able to deliver the fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI). To confirm gene silencing, a second set of primers was designed outside the dsRNA sequence of target gene. In this assay, the suppression of AKT and GSK transcripts (approximately 50% reduction in both genes) was demonstrated in 7-day-old eggs. Interestingly, silencing of GSK in 7-day-old eggs caused 25% reduction in hatching. Additionally, the effect of silencing AKT and GSK on embryo energy metabolism was evaluated. As expected, knockdown of AKT, which down regulates GSK, the suppressor of glycogen synthesis, decreased glycogen content in electroporated eggs. These data demonstrate that electroporation of de-waxed R. microplus eggs could be used for gene silencing in tick embryos, and improve the knowledge about arthropod embryogenesis. PMID:26091260

  9. Simple Arduino based pulse generator design for electroporation

    Science.gov (United States)

    Sulaeman, Muhammad Yangki; Widita, Rena

    2015-09-01

    This research will discuss the design of electroporation generator using Arduino as the pulse controller. The pulse parameters are the most important thing in electroporation method, therefore many researches aimed to produce generator to control its parameters easily. Arduino will be used as the microcontroller to create low amplitude signal trigger to get the high voltage pulse for electroporation. 124.4 VDC will be used and tested in cuvette contained NaCl solution with various concentration between 0% - 1%.

  10. Irreversible electroporation: state of the art.

    Science.gov (United States)

    Wagstaff, Peter Gk; Buijs, Mara; van den Bos, Willemien; de Bruin, Daniel M; Zondervan, Patricia J; de la Rosette, Jean Jmch; Laguna Pes, M Pilar

    2016-01-01

    The field of focal ablative therapy for the treatment of cancer is characterized by abundance of thermal ablative techniques that provide a minimally invasive treatment option in selected tumors. However, the unselective destruction inflicted by thermal ablation modalities can result in damage to vital structures in the vicinity of the tumor. Furthermore, the efficacy of thermal ablation intensity can be impaired due to thermal sink caused by large blood vessels in the proximity of the tumor. Irreversible electroporation (IRE) is a novel ablation modality based on the principle of electroporation or electropermeabilization, in which electric pulses are used to create nanoscale defects in the cell membrane. In theory, IRE has the potential of overcoming the aforementioned limitations of thermal ablation techniques. This review provides a description of the principle of IRE, combined with an overview of in vivo research performed to date in the liver, pancreas, kidney, and prostate.

  11. Nonviral Gene Therapy of the Nervous System: Electroporation.

    Science.gov (United States)

    Ding, Xue-Feng; Fan, Ming

    2016-01-01

    Electroporation has been widely used to efficiently transfer foreign genes into the mammalian central nervous system (CNS), and thus plays an important role in gene therapeutic studies on some brain disorders. A lot of work concerning electroporation is focused on gene transfer into rodent brains. This technique involves an injection of nucleic acids into the brain ventricle or specific area and then applying appropriate electrical field to the injected area. Here, we briefly introduced the advantages and the basic procedures of gene transfer into the rodent brain using electroporation. Better understanding of electroporation in rodent brain may further facilitate gene therapeutic studies on brain disorders.

  12. Electrical impedance tomographic imaging of a single cell electroporation.

    Science.gov (United States)

    Meir, Arie; Rubinsky, Boris

    2014-06-01

    A living cell placed in a high strength electric field, can undergo a process known as electroporation. It is believed that during electroporation nano-scale defects (pores) occur in the membrane of the cell, causing dramatic changes to the permeability of its membrane. Electroporation is an important technique in biotechnology and medicine and numerous methods are being developed to improve the understanding and use of the technology. We propose to extend the toolbox available for studying electroporation by generating impedance distribution images of the cell as it undergoes electroporation using Electrical Impedance Tomography (EIT). To investigate the feasibility of this concept, we develop a mathematical model of the process of electroporation in a single cell and of EIT of the process and show simulation results of a computer-based finite element model (FEM). Our work is an attempt to develop a new imaging tool for visualizing electroporation in a single cell, offering a different temporal and spatial resolution compared to the state of the art, which includes bulk measurements of electrical properties during single cell electroporation, patch clamp and voltage clamp measurement in single cells and optical imaging with colorimetric dyes during single cell electroporation. This paper is a preliminary theoretic feasibility study.

  13. Combining Electrolysis and Electroporation for Tissue Ablation.

    Science.gov (United States)

    Phillips, Mary; Rubinsky, Liel; Meir, Arie; Raju, Narayan; Rubinsky, Boris

    2015-08-01

    Electrolytic ablation is a method that operates by delivering low magnitude direct current to the target region over long periods of time, generating electrolytic products that destroy cells. This study was designed to explore the hypothesis stating that electrolytic ablation can be made more effective when the electrolysis-producing electric charges are delivered using electric pulses with field strength typical in reversible electroporation protocols. (For brevity we will refer to tissue ablation protocols that combine electroporation and electrolysis as E(2).) The mechanistic explanation of this hypothesis is related to the idea that products of electrolysis generated by E(2) protocols can gain access to the interior of the cell through the electroporation permeabilized cell membrane and therefore cause more effective cell death than from the exterior of an intact cell. The goal of this study is to provide a first-order examination of this hypothesis by comparing the charge dosage required to cause a comparable level of damage to a rat liver, in vivo, when using either conventional electrolysis or E(2) approaches. Our results show that E(2) protocols produce tissue damage that is consistent with electrolytic ablation. Furthermore, E(2) protocols cause damage comparable to that produced by conventional electrolytic protocols while delivering orders of magnitude less charge to the target tissue over much shorter periods of time.

  14. Foreign gene expression in an organotypic culture of cortical anlage after in vivo electroporation.

    Science.gov (United States)

    Miyasaka, N; Arimatsu, Y; Takiguchihayashi, K

    1999-08-01

    A high level of foreign gene expression in organotypic cultures of the cerebral cortical anlage was achieved by electroporation-mediated gene transfer in vivo. A mammalian expression plasmid for green fluorescent protein (GFP) gene was injected into the lateral ventricle of rat embryos. Immediately after the plasmid DNA injection, the head of the embryo was electroporated between a pair of tweezer-type electrodes. The cortical anlage was isolated and maintained organotypically up to 21 days in vitro (DIV). The GFP-transgene was expressed intensely in neural progenitor cells at 1 DIV. GFP-expressing cells were still detectable and were demonstrated to differentiate into neurons and glia at 21 DIV. This system is expected to be useful for molecular analysis of cerebral cortical development and function.

  15. Computer Based Optimisation Rutines

    DEFF Research Database (Denmark)

    Dragsted, Birgitte; Olsen, Flemmming Ove

    1996-01-01

    In this paper the need for optimisation methods for the laser cutting process has been identified as three different situations. Demands on the optimisation methods for these situations are presented, and one method for each situation is suggested. The adaptation and implementation of the methods...

  16. Computer Based Optimisation Rutines

    DEFF Research Database (Denmark)

    Dragsted, Birgitte; Olsen, Flemmming Ove

    1996-01-01

    In this paper the need for optimisation methods for the laser cutting process has been identified as three different situations. Demands on the optimisation methods for these situations are presented, and one method for each situation is suggested. The adaptation and implementation of the methods...

  17. Optimal optimisation in chemometrics

    NARCIS (Netherlands)

    Hageman, Joseph Albert

    2004-01-01

    The use of global optimisation methods is not straightforward, especially for the more difficult optimisation problems. Solutions have to be found for items such as the evaluation function, representation, step function and meta-parameters, before any useful results can be obtained. This thesis aims

  18. Optimized electroporation-induced transformation in Microcystis aeruginosa PCC7806

    Directory of Open Access Journals (Sweden)

    El Semary, A.

    2010-01-01

    Full Text Available Gene disruption in cyanobacteria is difficult and comprises an obstacle for genetic manipulation. Very few reports tackled this problem but the methods used are usually obscure and hardly reproducible. Here we describe an optimized electroporation-induced transformation in Microcystis aeruginosa PCC7806 where conditions for successful electroporation and transformation are investigated.

  19. Investigating cellular electroporation using planar membrane models and miniaturized devices

    NARCIS (Netherlands)

    Uitert, van Iris

    2010-01-01

    This thesis focuses on increasing our understanding of the electroporation process. Electroporation is a technique employed to introduce foreign molecules into cells that can normally not pass the cell membrane. By applying a short but high electric field, pores appear in the membrane through which

  20. Cell electroporation by CNT-featured microfluidic chip.

    Science.gov (United States)

    Shahini, Mehdi; Yeow, John T W

    2013-07-01

    We present the application of carbon nanotubes (CNTs) for cell electroporation that is performed in a microfluidic device. Lab on a chip (LOC) developments have raised unique possibilities to scale down cell manipulation systems to a cellular level to achieve higher performance and accuracy. Among the systems employed for cell disruption, electroporation without chemical reagents provides many advantages but suffers from high voltage requirements. We have exploited the electric field enhancement by CNTs to realize low-voltage electroporation. A microchip with embedded aligned CNTs has been developed to test the effect of the enhanced electric field on electroporation of mammalian CHO cells. Fluorogenic Calcein AM dye is used to image the release of the intercellular medium as an indication of electroporation. The electroporation phenomenon is recorded in real-time and compared with that of a device without CNTs. The results show that at a voltage as low as 3 volts, the electroporation yield rate is increased by 72% with the incorporation of CNTs. This enhancement is a promising advancement towards integration of low-voltage electroporation with other low-voltage cell manipulation techniques.

  1. Transfer of foreign DNA into aquatic animals by electroporation

    Science.gov (United States)

    This chapter describes the principle, procedure and application of the electroporation method to produce various types of transgenic marine organisms including finfish, shellfish and marine algae. Electroporation utilizes a series of short electrical pulses to induce formation of short-lived pores ...

  2. Electroporation into Cultured Mammalian Embryos

    Science.gov (United States)

    Nomura, Tadashi; Takahashi, Masanori; Osumi, Noriko

    Over the last century, mammalian embryos have been used extensively as a common animal model to investigate fundamental questions in the field of developmental biology. More recently, the establishment of transgenic and gene-targeting systems in laboratory mice has enabled researchers to unveil the genetic mechanisms under lying complex developmental processes (Mak, 2007). However, our understanding of cell—cell interactions and their molecular basis in the early stages of mammalian embryogenesis is still very fragmentary. One of the major problems is the difficulty of precise manipulation and limited accessibility to mammalian embryos via uterus wall. Unfortunately, existing tissue and organotypic culture systems per se do not fully recapitulate three-dimensional, dynamic processes of organogenesis observed in vivo. Although transgenic animal technology and virus-mediated gene delivery are useful to manipulate gene expression, these techniques take much time and financial costs, which limit their use.

  3. Tutorial: Electroporation of cells in complex materials and tissue

    Science.gov (United States)

    Rems, L.; Miklavčič, D.

    2016-05-01

    Electroporation is being successfully used in biology, medicine, food processing, and biotechnology, and in some environmental applications. Recent applications also include in addition to classical electroporation, where cells are exposed to micro- or milliseconds long pulses, exposures to extremely short nanosecond pulses, i.e., high-frequency electroporation. Electric pulses are applied to cells in different structural configurations ranging from suspended cells to cells in tissues. Understanding electroporation of cells in tissues and other complex environments is a key to its successful use and optimization in various applications. Thus, explanation will be provided theoretically/numerically with relation to experimental observations by scaling our understanding of electroporation from the molecular level of the cell membrane up to the tissue level.

  4. 脉冲电场介导AT2R基因在血管局部表达及其对血管新生内膜的影响%Electroporation-mediated angiotensin Ⅱ type 2 receptor expression improves neo intimal hyperplasia of carotid arteries in rats after balloon angioplasty

    Institute of Scientific and Technical Information of China (English)

    刘德峰; 屈小玲; 刘建平

    2011-01-01

    Objective To determine the effects of electroporation on the transfection of angiotensin Ⅱ type 2 receptor (AT2R) gene into rat carotid arteries and predict its role in neointimal hyperplasia of carotid arteries after balloon angioplasty. Methods After the establishment of rat carotid balloon injury restenosis model, 18 SD rats were divided into AT2R transfection group, empty plasmid transfection group (GFP), and non-transfection group (n = 6 ). AT2R gene plasmid or empty plasmid was transfected by electroporation into rat carotid arteries. Their arteries were harvested at 3, 14 and 21 d after gene transfer, with 2 rats at each time points. The expression of AT2B and morphology of the arteries were evaluated by immunohistochemistry and HE staining respoctively. Results Electroporation-mediated transfection of AT2R gene into injured rat carotid arteries significantly up-regulated the levels of AT2R mRNA in the neointima in a time dependent manner from day 3 to day 14 ( strong to moderate to mild expression). But no expression of AT2B was found in the other 2 groups at these 3 time points. At day 21, AT2R transfection significantly reduced intimal/medial area ratio as compared with non-transfection group and GFP transfection group (0.76 ± 0.08 vs 1.39 ± 0.08 and 1.32 ± 0. 10, P < 0.01 ). No significant difference was found between non-transfection group and GFP transfection group. Conclusion Our results indicate that electroporation is an effective means to introduce naked AT2R DNA into the blood vessel wall, and gene transfer of AT2R in vessel wall may effectively inhibit VSMC proliferation and neointimal hyperplasia in the rat carotid arteries after balloon angioplasty.%目的 研究脉冲电场对血管紧张素2型受体(AT2R)基因在血管局部表达的作用,探讨AT2R基因在体转染对大鼠颈动脉球囊损伤后新生内膜增生的作用.方法 大鼠颈动脉球囊损伤后,用脉冲电穿孔法介导AT2R cDNA真核表达质粒或空质粒载

  5. Isogeometric design optimisation

    NARCIS (Netherlands)

    Nagy, A.P.

    2011-01-01

    Design optimisation is of paramount importance in most engineering, e.g. aeronautical, automotive, or naval, disciplines. Its interdisciplinary character is manifested in the synthesis of geometric modelling, numerical analysis, mathematical programming, and computer sciences. The evolution of the f

  6. Efficient expression of transgenes in adult zebrafish by electroporation

    Directory of Open Access Journals (Sweden)

    Rao S Hari

    2005-10-01

    Full Text Available Abstract Background Expression of transgenes in muscle by injection of naked DNA is widely practiced. Application of electrical pulses at the site of injection was demonstrated to improve transgene expression in muscle tissue. Zebrafish is a precious model to investigate developmental biology in vertebrates. In this study we investigated the effect of electroporation on expression of transgenes in 3–6 month old adult zebrafish. Results Electroporation parameters such as number of pulses, voltage and amount of plasmid DNA were optimized and it was found that 6 pulses of 40 V·cm-1 at 15 μg of plasmid DNA per fish increased the luciferase expression 10-fold compared to controls. Similar enhancement in transgene expression was also observed in Indian carp (Labeo rohita. To establish the utility of adult zebrafish as a system for transient transfections, the strength of the promoters was compared in A2 cells and adult zebrafish after electroporation. The relative strengths of the promoters were found to be similar in cell lines and in adult zebrafish. GFP fluorescence in tissues after electroporation was also studied by fluorescence microscopy. Conclusion Electroporation after DNA injection enhances gene expression 10-fold in adult zebrafish. Electroporation parameters for optimum transfection of adult zebrafish with tweezer type electrode were presented. Enhanced reporter gene expression upon electroporation allowed comparison of strengths of the promoters in vivo in zebrafish.

  7. Synergistic Combination of Electrolysis and Electroporation for Tissue Ablation.

    Science.gov (United States)

    Stehling, Michael K; Guenther, Enric; Mikus, Paul; Klein, Nina; Rubinsky, Liel; Rubinsky, Boris

    2016-01-01

    Electrolysis, electrochemotherapy with reversible electroporation, nanosecond pulsed electric fields and irreversible electroporation are valuable non-thermal electricity based tissue ablation technologies. This paper reports results from the first large animal study of a new non-thermal tissue ablation technology that employs "Synergistic electrolysis and electroporation" (SEE). The goal of this pre-clinical study is to expand on earlier studies with small animals and use the pig liver to establish SEE treatment parameters of clinical utility. We examined two SEE methods. One of the methods employs multiple electrochemotherapy-type reversible electroporation magnitude pulses, designed in such a way that the charge delivered during the electroporation pulses generates the electrolytic products. The second SEE method combines the delivery of a small number of electrochemotherapy magnitude electroporation pulses with a low voltage electrolysis generating DC current in three different ways. We show that both methods can produce lesion with dimensions of clinical utility, without the need to inject drugs as in electrochemotherapy, faster than with conventional electrolysis and with lower electric fields than irreversible electroporation and nanosecond pulsed ablation.

  8. Plasmid DNA gene therapy by electroporation: principles and recent advances.

    Science.gov (United States)

    Murakami, Tatsufumi; Sunada, Yoshihide

    2011-12-01

    Simple plasmid DNA injection is a safe and feasible gene transfer method, but it confers low transfection efficiency and transgene expression. This non-viral gene transfer method is enhanced by physical delivery methods, such as electroporation and the use of a gene gun. In vivo electroporation has been rapidly developed over the last two decades to deliver DNA to various tissues or organs. It is generally considered that membrane permeabilization and DNA electrophoresis play important roles in electro-gene transfer. Skeletal muscle is a well characterized target tissue for electroporation, because it is accessible and allows for long-lasting gene expression ( > one year). Skin is also a target tissue because of its accessibility and immunogenicity. Numerous studies have been performed using in vivo electroporation in animal models of disease. Clinical trials of DNA vaccines and immunotherapy for cancer treatment using in vivo electroporation have been initiated in patients with melanoma and prostate cancer. Furthermore, electroporation has been applied to DNA vaccines for infectious diseases to enhance immunogenicity, and the relevant clinical trials have been initiated. The gene gun approach is also being applied for the delivery of DNA vaccines against infectious diseases to the skin. Here, we review recent advances in the mechanism of in vivo electroporation, and summarize the findings of recent preclinical and clinical studies using this technology.

  9. An update on irreversible electroporation of liver tumours.

    Science.gov (United States)

    Yeung, Enoch S L; Chung, Max W Y; Wong, Keedon; Wong, Clement Y K; So, Enoch C T; Chan, Albert C Y

    2014-08-01

    OBJECTIVE. To investigate the clinical efficacy and safety of irreversible electroporation for ablation of liver tumour in humans. DATA SOURCES. The PubMed and MEDLINE databases were systematically searched. STUDY SELECTION. Clinical research published in English in the last 10 years until October 2013 that address clinical issues related to irreversible electroporation of human liver tumours were selected. "Liver tumor", "local ablative therapy", and "irreversible electroporation" were used as the search terms. DATA EXTRACTION AND SYNTHESIS. The data extracted for this review was analysed by the authors, with a focus on the clinical efficacy and the safety of irreversible electroporation. The complete response rates look promising, ranging from 72% to 100%, except in one study in a subgroup of liver tumours in which the complete response rate was only 50% that was likely due to the inclusion of larger-size tumours. In one study, the local recurrence rate at 12 months was approximately 40%. As for the safety of irreversible electroporation, there were only a few reported complications (cardiac arrhythmia, pneumothorax, and electrolyte disturbance) that were mostly transient and not serious. There was no reported mortality related to the use of irreversible electroporation. CONCLUSION. Irreversible electroporation is a potentially effective liver tumour ablative therapy that gives rise to only mild and transient side-effects. Further studies with better patient selection criteria and longer follow-up are needed to clarify its role as a first-line liver tumour treatment modality.

  10. A nonlinear electromechanical coupling model for electropore expansion in cell electroporation

    Science.gov (United States)

    Deng, Peigang; Lee, Yi-Kuen; Zhang, Tong-Yi

    2014-11-01

    Under an electric field, the electric tractions acting on a cell membrane containing a pore-nucleus are investigated by using a nonlinear electromechanical coupling model, in which the cell membrane is treated as a hyperelastic material. Iterations between the electric field and the structure field are performed to reveal the electrical forces exerting on the pore region and the subsequent pore expansion process. An explicit exponential decay of the membrane’s edge energy as a function of pore radius is defined for a hydrophilic pore and the transition energy as a hydrophobic pore converts to a hydrophilic pore during the initial stage of pore formation is investigated. It is found that the edge energy for the creation of an electropore edge plays an important role at the atomistic scale and it determines the hydrophobic-hydrophilic transition energy barrier. Various free energy evolution paths are exhibited, depending on the applied electric field, which provides further insight towards the electroporation (EP) phenomenon. In comparison with previous EP models, the proposed model has the ability to predict the metastable point on the free energy curve that is relevant to the lipid ion channel. In addition, the proposed model can also predict the critical transmembrane potential for the activation of an effective electroporation that is in a good agreement with previously published experimental data.

  11. A nonlinear electromechanical coupling model for electropore expansion in cell electroporation

    KAUST Repository

    Deng, Peigang

    2014-10-15

    Under an electric field, the electric tractions acting on a cell membrane containing a pore-nucleus are investigated by using a nonlinear electromechanical coupling model, in which the cell membrane is treated as a hyperelastic material. Iterations between the electric field and the structure field are performed to reveal the electrical forces exerting on the pore region and the subsequent pore expansion process. An explicit exponential decay of the membrane\\'s edge energy as a function of pore radius is defined for a hydrophilic pore and the transition energy as a hydrophobic pore converts to a hydrophilic pore during the initial stage of pore formation is investigated. It is found that the edge energy for the creation of an electropore edge plays an important role at the atomistic scale and it determines the hydrophobic-hydrophilic transition energy barrier. Various free energy evolution paths are exhibited, depending on the applied electric field, which provides further insight towards the electroporation (EP) phenomenon. In comparison with previous EP models, the proposed model has the ability to predict the metastable point on the free energy curve that is relevant to the lipid ion channel. In addition, the proposed model can also predict the critical transmembrane potential for the activation of an effective electroporation that is in a good agreement with previously published experimental data.

  12. GFLV replication in electroporated grapevine protoplasts.

    Science.gov (United States)

    Valat; Toutain; Courtois; Gaire; Decout; Pinck; Mauro; Burrus

    2000-06-29

    Grapevine fanleaf virus (GFLV), responsible for the economically important court-noué disease, is exclusively transmitted to its natural host in the vineyards through Xiphinema nematodes. We have developed direct inoculation of GFLV into grapevine through protoplast electroporation. Protoplasts were isolated from mesophyll of in vitro-grown plants and from embryogenic cell suspensions. Permeation conditions were determined by monitoring calcein uptake. Low salt poration medium was selected. Electrical conditions leading to strong transient gene expression were also tested for GFLV inoculation (isolate F13). GFLV replication was detected with either virus particles (2 µg) or viral RNA (10 ng) in both protoplast populations, as shown by anti-P38 Western blotting. Direct inoculation and replication were also observed with Arabis mosaic virus (ArMV), a closely related nepovirus, as well as with another GFLV isolate. These results will be valuable in grapevine biotechnology, for GFLV replication studies, transgenic plant screening for GFLV resistance, and biorisk evaluation.

  13. Single cell electroporation using proton beam fabricated biochips

    Science.gov (United States)

    Homhuan, S.; Zhang, B.; Sheu, F.-S.; Bettiol, A. A.; Watt, F.

    2010-05-01

    We report the design and fabrication of a novel single cell electroporation biochip fabricated by the Proton Beam Writing technique (PBW), a new technique capable of direct-writing high-aspect-ratio nano and microstructures. The biochip features nickel micro-electrodes with straight-side walls between which individual cells are positioned. By applying electrical impulses across the electrodes, SYTOX® Green nucleic acid stain is incorporated into mouse neuroblastoma (N2a) cells. When the stain binds with DNA inside the cell nucleus, green fluorescence is observed upon excitation from a halogen lamp. Three parameters; electric field strength, pulse duration, and the number of pulses have been considered and optimized for the single cell electroporation. The results show that our biochip gives successfully electroporated cells . This single cell electroporation system represents a promising method for investigating the introduction of a wide variety of fluorophores, nanoparticles, quantum dots, DNAs and proteins into cells.

  14. Pulsed Electromagnetic Field Assisted in vitro Electroporation: A Pilot Study

    Science.gov (United States)

    Novickij, Vitalij; Grainys, Audrius; Lastauskienė, Eglė; Kananavičiūtė, Rūta; Pamedytytė, Dovilė; Kalėdienė, Lilija; Novickij, Jurij; Miklavčič, Damijan

    2016-09-01

    Electroporation is a phenomenon occurring due to exposure of cells to Pulsed Electric Fields (PEF) which leads to increase of membrane permeability. Electroporation is used in medicine, biotechnology, and food processing. Recently, as an alternative to electroporation by PEF, Pulsed ElectroMagnetic Fields (PEMF) application causing similar biological effects was suggested. Since induced electric field in PEMF however is 2–3 magnitudes lower than in PEF electroporation, the membrane permeabilization mechanism remains hypothetical. We have designed pilot experiments where Saccharomyces cerevisiae and Candida lusitaniae cells were subjected to single 100–250 μs electrical pulse of 800 V with and without concomitant delivery of magnetic pulse (3, 6 and 9 T). As expected, after the PEF pulses only the number of Propidium Iodide (PI) fluorescent cells has increased, indicative of membrane permeabilization. We further show that single sub-millisecond magnetic field pulse did not cause detectable poration of yeast. Concomitant exposure of cells to pulsed electric (PEF) and magnetic field (PMF) however resulted in the increased number PI fluorescent cells and reduced viability. Our results show increased membrane permeability by PEF when combined with magnetic field pulse, which can explain electroporation at considerably lower electric field strengths induced by PEMF compared to classical electroporation.

  15. Thermal loading in flow-through electroporation microfluidic devices.

    Science.gov (United States)

    del Rosal, Blanca; Sun, Chen; Loufakis, Despina Nelie; Lu, Chang; Jaque, Daniel

    2013-08-01

    Thermal loading effects in flow-through electroporation microfluidic devices have been systematically investigated by using dye-based ratiometric luminescence thermometry. Fluorescence measurements have revealed the crucial role played by both the applied electric field and flow rate on the induced temperature increments at the electroporation sections of the devices. It has been found that Joule heating could raise the intra-channel temperature up to cytotoxic levels (>45 °C) only when conditions of low flow rates and high applied voltages are applied. Nevertheless, when flow rates and electric fields are set to those used in real electroporation experiments we have found that local heating is not larger than a few degrees, i.e. temperature is kept within the safe range (electroporation devices from which the heat affected area can be elucidated. Experimental data have been found to be in excellent agreement with numerical simulations that have also revealed the presence of a non-homogeneous temperature distribution along the electroporation channel whose magnitude is critically dependent on both applied electric field and flow rate. Results included in this work will allow for full control over the electroporation conditions in flow-through microfluidic devices.

  16. Simulation versus Optimisation

    DEFF Research Database (Denmark)

    Lund, Henrik; Arler, Finn; Østergaard, Poul Alberg

    2017-01-01

    investment optimisation or optimal solutions approach. On the other hand the analytical simulation or alternatives assessment approach. Awareness of the dissimilar theoretical assumption behind the models clarifies differences between the models, explains dissimilarities in results, and provides...... a theoretical and methodological foundation for understanding and interpreting results from the two archetypes. Keywords: energy system analysis; investment optimisation models; simulations models; modelling theory;renewable energy......In recent years, several tools and models have been developed and used for the design and analysis of future national energy systems. Many of these models focus on the integration of various renewable energy resources and the transformation of existing fossil-based energy systems into future...

  17. Optimising AspectJ

    DEFF Research Database (Denmark)

    Avgustinov, Pavel; Christensen, Aske Simon; Hendren, Laurie

    2005-01-01

    AspectJ, an aspect-oriented extension of Java, is becoming increasingly popular. However, not much work has been directed at optimising compilers for AspectJ. Optimising AOP languages provides many new and interesting challenges for compiler writers, and this paper identifies and addresses three...... all of the techniques in this paper in abc, our AspectBench Compiler for AspectJ, and we demonstrate significant speedups with empirical results. Some of our techniques have already been integrated into the production AspectJ compiler, ajc 1.2.1....

  18. Modeling of electric field distribution in tissues during electroporation.

    Science.gov (United States)

    Corovic, Selma; Lackovic, Igor; Sustaric, Primoz; Sustar, Tomaz; Rodic, Tomaz; Miklavcic, Damijan

    2013-02-21

    Electroporation based therapies and treatments (e.g. electrochemotherapy, gene electrotransfer for gene therapy and DNA vaccination, tissue ablation with irreversible electroporation and transdermal drug delivery) require a precise prediction of the therapy or treatment outcome by a personalized treatment planning procedure. Numerical modeling of local electric field distribution within electroporated tissues has become an important tool in treatment planning procedure in both clinical and experimental settings. Recent studies have reported that the uncertainties in electrical properties (i.e. electric conductivity of the treated tissues and the rate of increase in electric conductivity due to electroporation) predefined in numerical models have large effect on electroporation based therapy and treatment effectiveness. The aim of our study was to investigate whether the increase in electric conductivity of tissues needs to be taken into account when modeling tissue response to the electroporation pulses and how it affects the local electric distribution within electroporated tissues. We built 3D numerical models for single tissue (one type of tissue, e.g. liver) and composite tissue (several types of tissues, e.g. subcutaneous tumor). Our computer simulations were performed by using three different modeling approaches that are based on finite element method: inverse analysis, nonlinear parametric and sequential analysis. We compared linear (i.e. tissue conductivity is constant) model and non-linear (i.e. tissue conductivity is electric field dependent) model. By calculating goodness of fit measure we compared the results of our numerical simulations to the results of in vivo measurements. The results of our study show that the nonlinear models (i.e. tissue conductivity is electric field dependent: σ(E)) fit experimental data better than linear models (i.e. tissue conductivity is constant). This was found for both single tissue and composite tissue. Our results of

  19. Optimising Magnetostatic Assemblies

    DEFF Research Database (Denmark)

    Insinga, Andrea Roberto; Smith, Anders

    the optimal remanence distribution with respect to a linear objective functional. Additionally, it is shown here that the same formalism can be applied to the optimisation of the geometry of magnetic systems. Specifically, the border separating the permanent magnet from regions occupied by air or soft...

  20. A theoretical analysis of the feasibility of a singularity-induced micro-electroporation system.

    Directory of Open Access Journals (Sweden)

    Gregory D Troszak

    Full Text Available Electroporation, the permeabilization of the cell membrane lipid bilayer due to a pulsed electric field, has important implications in the biotechnology, medicine, and food industries. Traditional macro and micro-electroporation devices have facing electrodes, and require significant potential differences to induce electroporation. The goal of this theoretical study is to investigate the feasibility of singularity-induced micro-electroporation; an electroporation configuration aimed at minimizing the potential differences required to induce electroporation by separating adjacent electrodes with a nanometer-scale insulator. In particular, this study aims to understand the effect of (1 insulator thickness and (2 electrode kinetics on electric field distributions in the singularity-induced micro-electroporation configuration. A non-dimensional primary current distribution model of the micro-electroporation channel shows that while increasing insulator thickness results in smaller electric field magnitudes, electroporation can still be performed with insulators thick enough to be made with microfabrication techniques. Furthermore, a secondary current distribution model of the singularity-induced micro-electroporation configuration with inert platinum electrodes and water electrolyte indicates that electrode kinetics do not inhibit charge transfer to the extent that prohibitively large potential differences are required to perform electroporation. These results indicate that singularity-induced micro-electroporation could be used to develop an electroporation system that consumes minimal power, making it suitable for remote applications such as the sterilization of water and other liquids.

  1. The Effects of Metallic Implants on Electroporation Therapies: Feasibility of Irreversible Electroporation for Brachytherapy Salvage

    Energy Technology Data Exchange (ETDEWEB)

    Neal, Robert E., E-mail: robert.neal@alfred.org.au [The Alfred Hospital, Radiology Research Unit, Department of Radiology (Australia); Smith, Ryan L., E-mail: ryan.smith@wbrc.org.au [The Alfred Hospital, William Buckland Radiotherapy Centre (Australia); Kavnoudias, Helen, E-mail: H.Kavnoudias@alfred.org.au [The Alfred Hospital, Radiology Research Unit, Department of Radiology (Australia); Rosenfeldt, Franklin, E-mail: F.Rosenfeldt@alfred.org.au; Ou, Ruchong, E-mail: Ruchong.Ou@bakeridi.edu.au [Monash University, Department of Surgery (Australia); Mclean, Catriona A., E-mail: C.Mclean@alfred.org.au [The Alfred Hospital, Department of Anatomical Pathology (Australia); Davalos, Rafael V., E-mail: davalos@vt.edu [Virginia Tech, School of Biomedical Engineering and Sciences (United States); Thomson, Kenneth R., E-mail: K.Thomson@alfred.org.au [The Alfred Hospital, Radiology Research Unit, Department of Radiology (Australia)

    2013-12-15

    Purpose: Electroporation-based therapies deliver brief electric pulses into a targeted volume to destabilize cellular membranes. Nonthermal irreversible electroporation (IRE) provides focal ablation with effects dependent on the electric field distribution, which changes in heterogeneous environments. It should be determined if highly conductive metallic implants in targeted regions, such as radiotherapy brachytherapy seeds in prostate tissue, will alter treatment outcomes. Theoretical and experimental models determine the impact of prostate brachytherapy seeds on IRE treatments. Materials and Methods: This study delivered IRE pulses in nonanimal, as well as in ex vivo and in vivo tissue, with and in the absence of expired radiotherapy seeds. Electrical current was measured and lesion dimensions were examined macroscopically and with magnetic resonance imaging. Finite-element treatment simulations predicted the effects of brachytherapy seeds in the targeted region on electrical current, electric field, and temperature distributions. Results: There was no significant difference in electrical behavior in tissue containing a grid of expired radiotherapy seeds relative to those without seeds for nonanimal, ex vivo, and in vivo experiments (all p > 0.1). Numerical simulations predict no significant alteration of electric field or thermal effects (all p > 0.1). Histology showed cellular necrosis in the region near the electrodes and seeds within the ablation region; however, there were no seeds beyond the ablation margins. Conclusion: This study suggests that electroporation therapies can be implemented in regions containing small metallic implants without significant changes to electrical and thermal effects relative to use in tissue without the implants. This supports the ability to use IRE as a salvage therapy option for brachytherapy.

  2. Recent Trends on Micro/Nanofluidic Single Cell Electroporation

    Directory of Open Access Journals (Sweden)

    Tuhin Subhra Santra

    2013-09-01

    Full Text Available The behaviors of cell to cell or cell to environment with their organelles and their intracellular physical or biochemical effects are still not fully understood. Analyzing millions of cells together cannot provide detailed information, such as cell proliferation, differentiation or different responses to external stimuli and intracellular reaction. Thus, single cell level research is becoming a pioneering research area that unveils the interaction details in high temporal and spatial resolution among cells. To analyze the cellular function, single cell electroporation can be conducted by employing a miniaturized device, whose dimension should be similar to that of a single cell. Micro/nanofluidic devices can fulfill this requirement for single cell electroporation. This device is not only useful for cell lysis, cell to cell fusion or separation, insertion of drug, DNA and antibodies inside single cell, but also it can control biochemical, electrical and mechanical parameters using electroporation technique. This device provides better performance such as high transfection efficiency, high cell viability, lower Joule heating effect, less sample contamination, lower toxicity during electroporation experiment when compared to bulk electroporation process. In addition, single organelles within a cell can be analyzed selectively by reducing the electrode size and gap at nanoscale level. This advanced technique can deliver (in/out biomolecules precisely through a small membrane area (micro to nanoscale area of the single cell, known as localized single cell membrane electroporation (LSCMEP. These articles emphasize the recent progress in micro/nanofluidic single cell electroporation, which is potentially beneficial for high-efficient therapeutic and delivery applications or understanding cell to cell interaction.

  3. Gold nanoparticles enhanced electroporation for mammalian cell transfection.

    Science.gov (United States)

    Zu, Yingbo; Huang, Shuyan; Liao, Wei-Ching; Lu, Yang; Wang, Shengnian

    2014-06-01

    Electroporation figured prominently as an effective nonviral gene delivery approach for its balance on the transfection efficiency and cell viability, no restrictions of probe or cell type, and operation simplicity. The commercial electroporation systems have been widely adopted in the past two decades while still carry drawbacks associated with the high applied electric voltage, unsatisfied delivery efficiency, and/or low cell viability. By adding highly conductive gold nanoparticles (AuNPs) in electroporation solution, we demonstrated enhanced electroporation performance (i.e., better DNA delivery efficiency and higher cell viability) on mammalian cells from two different aspects: the free, naked AuNPs reduce the resistance of the electroporation solution so that the local pulse strength on cells was enhanced; targeting AuNPs (e.g., Tf-AuNPs) were brought to the cell membrane to work as virtual microelectrodes to porate cells with limited area from many different sites. The enhancement was confirmed with leukemia cells in both a commercial batch electroporation system and a home-made flow-through system using pWizGFP plasmid DNA probes. Such enhancement depends on the size, concentration, and the mixing ratio of free AuNPs/Tf-AuNPs. An equivalent mixture of free AuNPs and Tf-AuNPs exhibited the best enhancement with the transfection efficiency increased 2-3 folds at minimum sacrifice of cell viability. This new delivery concept, the combination of nanoparticles and electroporation technologies, may stimulate various in vitro and in vivo biomedical applications which rely on the efficient delivery of nucleic acids, anticancer drugs, or other therapeutic materials.

  4. High-throughput in situ cell electroporation microsystem for parallel delivery of single guide RNAs into mammalian cells

    Science.gov (United States)

    Bian, Shengtai; Zhou, Yicen; Hu, Yawei; Cheng, Jing; Chen, Xiaofang; Xu, Youchun; Liu, Peng

    2017-01-01

    Arrayed genetic screens mediated by the CRISPR/Cas9 technology with single guide RNA (sgRNA) libraries demand a high-throughput platform capable of transfecting diverse cell types at a high efficiency in a genome-wide scale for detection and analysis of sophisticated cellular phenotypes. Here we developed a high-throughput in situ cell electroporation (HiCEP) microsystem which leveraged the superhydrophobic feature of the microwell array to achieve individually controlled conditions in each microwell and coupled an interdigital electrode array chip with the microwells in a modular-based scheme for highly efficient delivery of exogenous molecules into cells. Two plasmids encoding enhanced green and red fluorescent proteins (EGFP and ERFP), respectively, were successfully electroporated into attached HeLa cells on a 169-microwell array chip with transfection efficiencies of 71.6 ± 11.4% and 62.9 ± 2.7%, and a cell viability above 95%. We also successfully conducted selective electroporation of sgRNA into 293T cells expressing the Cas9 nuclease in a high-throughput manner and observed the four-fold increase of the GFP intensities due to the repair of the protein coding sequences mediated by the CRISPR/Cas9 system. This study proved that this HiCEP system has the great potential to be used for arrayed functional screens with genome-wide CRISPR libraries on hard-to-transfect cells in the future. PMID:28211892

  5. Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes.

    Science.gov (United States)

    Chen, Sean; Lee, Benjamin; Lee, Angus Yiu-Fai; Modzelewski, Andrew J; He, Lin

    2016-07-08

    The CRISPR/Cas9 system has been employed to efficiently edit the genomes of diverse model organisms. CRISPR-mediated mouse genome editing is typically accomplished by microinjection of Cas9 DNA/RNA and single guide RNA (sgRNA) into zygotes to generate modified animals in one step. However, microinjection is a technically demanding, labor-intensive, and costly procedure with poor embryo viability. Here, we describe a simple and economic electroporation-based strategy to deliver Cas9/sgRNA ribonucleoproteins into mouse zygotes with 100% efficiency for in vivo genome editing. Our methodology, designated as CRISPR RNP Electroporation of Zygotes (CRISPR-EZ), enables highly efficient and high-throughput genome editing in vivo, with a significant improvement in embryo viability compared with microinjection. Using CRISPR-EZ, we generated a variety of editing schemes in mouse embryos, including indel (insertion/deletion) mutations, point mutations, large deletions, and small insertions. In a proof-of-principle experiment, we used CRISPR-EZ to target the tyrosinase (Tyr) gene, achieving 88% bi-allelic editing and 42% homology-directed repair-mediated precise sequence modification in live mice. Taken together, CRISPR-EZ is simple, economic, high throughput, and highly efficient with the potential to replace microinjection for in vivo genome editing in mice and possibly in other mammals.

  6. Application of Electroporation Technique in Biofuel Processing

    Directory of Open Access Journals (Sweden)

    Yousuf Abu

    2017-01-01

    Full Text Available Biofuels production is mostly oriented with fermentation process, which requires fermentable sugar as nutrient for microbial growth. Lignocellulosic biomass (LCB represents the most attractive, low-cost feedstock for biofuel production, it is now arousing great interest. The cellulose that is embedded in the lignin matrix has an insoluble, highly-crystalline structure, so it is difficult to hydrolyze into fermentable sugar or cell protein. On the other hand, microbial lipid has been studying as substitute of plant oils or animal fat to produce biodiesel. It is still a great challenge to extract maximum lipid from microbial cells (yeast, fungi, algae investing minimum energy.Electroporation (EP of LCB results a significant increase in cell conductivity and permeability caused due to the application of an external electric field. EP is required to alter the size and structure of the biomass, to reduce the cellulose crystallinity, and increase their porosity as well as chemical composition, so that the hydrolysis of the carbohydrate fraction to monomeric sugars can be achieved rapidly and with greater yields. Furthermore, EP has a great potential to disrupt the microbial cell walls within few seconds to bring out the intracellular materials (lipid to the solution. Therefore, this study aims to describe the challenges and prospect of application of EP technique in biofuels processing.

  7. Irreversible Electroporation for Colorectal Liver Metastases.

    Science.gov (United States)

    Scheffer, Hester J; Melenhorst, Marleen C A M; Echenique, Ana M; Nielsen, Karin; van Tilborg, Aukje A J M; van den Bos, Willemien; Vroomen, Laurien G P H; van den Tol, Petrousjka M P; Meijerink, Martijn R

    2015-09-01

    Image-guided tumor ablation techniques have significantly broadened the treatment possibilities for primary and secondary hepatic malignancies. A new ablation technique, irreversible electroporation (IRE), was recently added to the treatment armamentarium. As opposed to thermal ablation, cell death with IRE is primarily induced using electrical energy: electrical pulses disrupt the cellular membrane integrity, resulting in cell death while sparing the extracellular matrix of sensitive structures such as the bile ducts, blood vessels, and bowel wall. The preservation of these structures makes IRE attractive for colorectal liver metastases (CRLM) that are unsuitable for resection and thermal ablation owing to their anatomical location. This review discusses different technical and practical issues of IRE for CRLM: the indications, patient preparations, procedural steps, and different "tricks of the trade" used to improve safety and efficacy of IRE. Imaging characteristics and early efficacy results are presented. Much is still unknown about the exact mechanism of cell death and about factors playing a crucial role in the extent of cell death. At this time, IRE for CRLM should only be reserved for small tumors that are truly unsuitable for resection or thermal ablation because of abutment of the portal triad or the venous pedicles.

  8. Online bioimpedance feedback for in vivo electroporated tissues

    Science.gov (United States)

    Medrano, J.; Rey, J. I.; Connolly, R. J.; Anderson, A.; Jaroszeski, M.; Gitlin, R.

    2010-04-01

    Electroporation in vivo is a biotechnology method that uses short-duration high intensity electric fields to enhance plasma membrane permeability in living cells in order to facilitate the uptake of drugs, DNA, genes and proteins into the cytoplasm. The degree of permeability is related to the tissue's bioimpedance; hence, accurate impedance evaluation throughout electroporation treatment is essential to 1) avoid over-treating tissues resulting in excessive cell death and 2) under-treating tissues resulting in poor permeability. Cell viability and membrane permeability is based on a number of factors, including: time elapsed after electroporation, electroporation pulse amplitude, tissue type, and so on; thus, efficient feedback protocols must minimize delays between treatment and impedance readings. Current methods of bioimpedance feedback are often cumbersome and impedance analysis devices can be expensive, bulky, and immobile. Emerging technologies facilitate economical methods, fast protocols, and portability to realize bioimpedance measurement and feedback online (i.e. realtime). Consequently, this research uses automation software, logic-biased protocols, an inexpensive commercially available impedance analyzer microchip, and a custom-built hexagonal electrode probe to measure dynamic bioimpedance changes. This work demonstrates how this novel system measures tissue bioimpedance instantly and efficiently before and after electroporation. Additionally this system allows for the comparison of electrode geometries as well as electric field' magnitudes and distributions. Follow up work will pursue the optimization of plasma membrane permeability for several tissue/cell types.

  9. The influence of soft layer electrokinetics on bacterial electroporation

    Science.gov (United States)

    Moran, Jeffrey; Dingari, Naga Neehar; Buie, Cullen

    2015-11-01

    Electroporation of mammalian cells has received a significant amount of theoretical attention over the last decade because of its ability to deliver biologically active molecules into cells using short and strong electric field pulses. However, application of the same theory to bacterial electroporation presents significant challenges because of the presence of charged soft layers around bacteria. The soft layer charge distribution has been found to significantly influence bacterial electrophoretic mobility and polarizability because it alters the electric potential spatial distribution around the cell envelope. In addition, the RC charging time scale of both the soft layer and electric double layer is of the order of microseconds, which is also of similar order of magnitude as the pore creation time scale. Therefore in this study, we investigate the influence of soft layer electrokinetics on the spatial pore distribution and the temporal pore radius evolution during bacteria electroporation, which are quantitative measures of a bacterium's amenability to electroporation. The study will have significant impact on designing and optimizing bacteria electroporation platforms for gene and drug delivery applications.

  10. Optimisation by hierarchical search

    Science.gov (United States)

    Zintchenko, Ilia; Hastings, Matthew; Troyer, Matthias

    2015-03-01

    Finding optimal values for a set of variables relative to a cost function gives rise to some of the hardest problems in physics, computer science and applied mathematics. Although often very simple in their formulation, these problems have a complex cost function landscape which prevents currently known algorithms from efficiently finding the global optimum. Countless techniques have been proposed to partially circumvent this problem, but an efficient method is yet to be found. We present a heuristic, general purpose approach to potentially improve the performance of conventional algorithms or special purpose hardware devices by optimising groups of variables in a hierarchical way. We apply this approach to problems in combinatorial optimisation, machine learning and other fields.

  11. Development of an efficient electroporation method for rhizobacterial Bacillus mycoides strains

    NARCIS (Netherlands)

    Yi, Yanglei; Kuipers, Oscar P

    2016-01-01

    In order to develop a method for electroporation of environmental Bacillus mycoides strains, we optimized several conditions that affect the electroporation efficiency of this bacterium. By combining the optimized conditions, the electroporation efficiency of strain EC18 was improved to (1.3 ± 0.6)

  12. Transient gene expression in electroporated intact tissues of Stylosanthes guianensis (Aubl.) Sw.

    OpenAIRE

    Quecini Vera Maria; Vieira Maria Lúcia Carneiro

    2001-01-01

    Genetic transformation though protoplast electroporation has been established for commercially important plant species. In this work, explant sources, electric field strengths, electroporation buffers, DNA forms and osmotic pretreatment were assayed in order to optimize transient reporter gene expression in electroporated tissues of Stylosanthes guianensis, a tropical forage legume. Higher transformation rates were obtained employing cotyledonary explants and an electric field strength of 250...

  13. Low-frequency ac electroporation shows strong frequency dependence and yields comparable transfection results to dc electroporation.

    Science.gov (United States)

    Zhan, Yihong; Cao, Zhenning; Bao, Ning; Li, Jianbo; Wang, Jun; Geng, Tao; Lin, Hao; Lu, Chang

    2012-06-28

    Conventional electroporation has been conducted by employing short direct current (dc) pulses for delivery of macromolecules such as DNA into cells. The use of alternating current (ac) field for electroporation has mostly been explored in the frequency range of 10kHz-1MHz. Based on Schwan equation, it was thought that with low ac frequencies (10Hz-10kHz), the transmembrane potential does not vary with the frequency. In this report, we utilized a flow-through electroporation technique that employed continuous 10Hz-10kHz ac field (based on either sine waves or square waves) for electroporation of cells with defined duration and intensity. Our results reveal that electropermeabilization becomes weaker with increased frequency in this range. In contrast, transfection efficiency with DNA reaches its maximum at medium frequencies (100-1000Hz) in the range. We postulate that the relationship between the transfection efficiency and the ac frequency is determined by combined effects from electrophoretic movement of DNA in the ac field, dependence of the DNA/membrane interaction on the ac frequency, and variation of transfection under different electropermeabilization intensities. The fact that ac electroporation in this frequency range yields high efficiency for transfection (up to ~71% for Chinese hamster ovary cells) and permeabilization suggests its potential for gene delivery.

  14. Permeabilizing soybean protoplasts to macromolecules using electroporation and hypotonic shock.

    Science.gov (United States)

    Cutler, A J; Saleem, M

    1987-01-01

    The percentage of soybean cell culture protoplasts permeabilized by electroporation was dependent on the voltage and the number of successive pulses that were applied. Best results were obtained with two 50 milliseconds, 400 volts per centimeter pulses after which 78% of the surviving protoplasts had been permeabilized to the fluorescent dye calcein. Quantitation of the volume of extracellular fluid taken up was performed using radioactive inulin (molecular weight 5000-5500). Typically between 20 and 40 nanoliters of fluid was taken up by 10(6) protoplasts. Electroporation and hypotonic shock treatments (M Saleem, AJ Cutler 1986 J Plant Physiol 124: 11-21) were compared with respect to the volume of fluid taken up under optimum conditions. Electroporation produced 10 times more uptake than hypotonic shock treatment. In all experiments there was a direct relationship between the number of protoplasts lysed and both the amount of fluid taken up and the percentage of surviving protoplasts that were permeabilized.

  15. Simple Genome Editing of Rodent Intact Embryos by Electroporation.

    Directory of Open Access Journals (Sweden)

    Takehito Kaneko

    Full Text Available The clustered regularly interspaced short palindromic repeat (CRISPR/CRISPR-associated (Cas system is a powerful tool for genome editing in animals. Recently, new technology has been developed to genetically modify animals without using highly skilled techniques, such as pronuclear microinjection of endonucleases. Technique for animal knockout system by electroporation (TAKE method is a simple and effective technology that produces knockout rats by introducing endonuclease mRNAs into intact embryos using electroporation. Using TAKE method and CRISPR/Cas system, the present study successfully produced knockout and knock-in mice and rats. The mice and rats derived from embryos electroporated with Cas9 mRNA, gRNA and single-stranded oligodeoxynucleotide (ssODN comprised the edited targeted gene as a knockout (67% of mice and 88% of rats or knock-in (both 33%. The TAKE method could be widely used as a powerful tool to produce genetically modified animals by genome editing.

  16. Gold nanoparticle-enhanced electroporation for leukemia cell transfection.

    Science.gov (United States)

    Huang, Shuyan; Zu, Yingbo; Wang, Shengnian

    2014-01-01

    Electroporation serves as an attractive nonviral gene delivery approach for its effectiveness, operational simplicity, and no restrictions of probe or cell type. The commercial electroporation systems have been widely adopted in research and clinics with protocols usually compromising appropriate transfection efficiency and cell viability. By introducing gold nanoparticles (AuNPs), we demonstrated greatly enhanced performance of electroporation from two aspects: the highly conductive, naked AuNPs help reduce the potential drop consumed by the electroporation solution so that the majority of the applied voltage of an electric pulse is truly imposed on cells with enhanced field strength; AuNPs with targeting ligands (e.g., transferrin-AuNPs or Tf-AuNPs) are bound to the cell membrane, working as virtual microelectrodes to create pores on cells with limited opening area while from many different sites. The addition of AuNPs during electroporation therefore benefits not only quicker recovery and better survival of cells but also more efficient uptake of the subjected probes. Such enhancement was successfully confirmed on a chronic myeloid leukemia cell line (i.e., K562 cells) in both a commercial batch electroporation system and a homemade flow system with pWizGFP plasmid DNA probes. The efficiency was found to be dependent on the size, concentration, and mixing ratio of free AuNPs/Tf-AuNPs. An equivalent mixture of free AuNPs and Tf-AuNPs exhibited the best enhancement with the transfection efficiency increase of two to threefold at a minimum sacrifice of the cell viability.

  17. Direct analysis of RNA transcripts in electroporated carrot protoplasts.

    Science.gov (United States)

    Murray, E E; Buchholz, W G; Bowen, B

    1990-07-01

    We describe a method for direct analysis of RNA transcribed from DNA introduced into carrot cells by electroporation. Octopine synthase RNA transcribed from the plasmid p35SOcs was detected in total and poly A(+) RNA on Northern blots and in RNA protection assays. The highest level of octopine synthase transcript was detected at approximately 8 hrs post-electroporation, although RNA could still be detected after 48 hrs. This method allows detection of foreign gene expression in a plant system and bypasses the need for reporter genes.

  18. Clinical study on safety and immunogenicity of therapeutic dual-plasmid HBV DNA vaccine mediated by in vivo electroporation%电脉冲介导的治疗性双质粒HBV DNA疫苗的临床安全性及免疫原性研究

    Institute of Scientific and Technical Information of China (English)

    杨海燕; 陈光明; 崔一民; 赵侠; 梅川; 饶桂荣; 莫国玉; 杨若才; 杨富强

    2013-01-01

    目的 观察电脉冲(EP)介导的治疗性双质粒HBV DNA疫苗的安全性及免疫原性.方法 将30名健康志愿者随机分为低(1mg)、中(2mg)、高(4mg)三个剂量组(n=10).于0、4、12、24周肌注联合EP导入治疗性双质粒HBVDNA疫苗.每个剂量组随机再分成两组(n=5),分别使用两种不同输出电压(36V和60V)导入DNA疫苗.观察受试者HBVDNA疫苗给药前后生命体征,物理学诊断指标(心电图、胸透、B超),实验室检查指标(血、尿常规、血液生化、凝血酶原时间、甲状腺功能、肿瘤标记物),免疫学检测指标[干扰素y(IFN-γ)、抗核抗体(ANA)、抗双链DNA抗体]、HBV血清标记物(HBsAg、HBcAb、HBeAg、HBeAb、HBV DNA)及抗HBs等的变化.结果 所有志愿者接受疫苗后,耐受性良好、生命体征平稳,个别受试者出现一过性体检指标升高或轻度异常,怀疑与用药有关,但均能自行缓解或恢复;抗HBs在大剂量36V组中有升高趋势,其中1例受试者给药后达17.22mU/ml.结论 EP介导的治疗性双质粒HBVDNA疫苗在低、中、高三个剂量组中均显示较好的耐受性和安全性,且在大剂量组中具有一定的体液免疫原性.%Objective To evaluate the safety and immunogenicity of the therapeutic dual-plasmid HBV DNA vaccine mediated by electroporation (EP) in vivo against the hepatitis B virus in healthy adult volunteers. Methods The enrolled 30 healthy volunteers were randomly divided into three dosage groups (10 volunteers in each group), namely: high-dose (4mg), middle-dose (2mg) and low-dose (1mg) groups. Volunteers received four intramuscular injections of HBV DNA vaccine mediated by in vivo EP at the 0, 4th, 12th and 24th week. Each dose group was further divided into 2 sub-groups (5 persons/per group) with different EP frequencies, i.e. 36 and 60 volt. The changes in response was determined by physical diagnosis (ECG, chest X-ray, type-B ultrasound), lab findings (blood and urine routine, blood

  19. Continuous cell electroporation for efficient DNA and siRNA delivery based on laminar microfluidic chips.

    Science.gov (United States)

    Wei, Zewen; Li, Zhihong

    2014-01-01

    Electroporation is a high-efficiency and low-toxicity physical gene transfer method. Traditional electroporation is limited to only low volume cell samples. Here we present a continuous cell electroporation method based on commonly used microfluidic chip fabrication technology. Using easily fabricated PDMS microfluidic chip, syringe pumps, and pulse generator, we show efficient delivery of both DNA and siRNA into different cell lines. We describe the protocol of chip fabrication, apparatus setup, and cell electroporation assay. Typically, the fabrication of the devices takes 1 or 2 days and the continuous electroporation assay takes 1 h.

  20. Electroporation of the hindbrain to trace axonal trajectories and synaptic targets in the chick embryo.

    Science.gov (United States)

    Kohl, Ayelet; Hadas, Yoav; Klar, Avihu; Sela-Donenfeld, Dalit

    2013-05-29

    Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

  1. Electrodiffusion of molecules in aqueous media: a robust, discretized description for electroporation and other transport phenomena.

    Science.gov (United States)

    Smith, Kyle C; Weaver, James C

    2012-06-01

    Electrically driven transport of molecules and ions within aqueous electrolytes is of long-standing interest, with direct relevance to applications that include the delivery/release of biologically active solutes to/from cells and tissues. Examples include iontophoretic and electroporation-mediated drug delivery. Here, we describe a robust method for characterizing electrodiffusive transport in physiologic aqueous media. Specifically, we treat the case of solute present in sufficiently low concentration as to negligibly contribute to the total ionic current within the system. In this limiting case, which applies to many systems of interest, the predominant electrical behavior due to small ions is decoupled from solute transport. Thus, electrical behavior may be characterized using existing methods and treated as known in characterizing electrodiffusive molecular transport. First, we present traditional continuum equations governing electrodiffusion of charged solutes within aqueous electrolytes and then adapt them to discretized systems. Second, we examine the time-dependent and steady-state interfacial concentration gradients that result from the combination of diffusion and electrical drift. Third, we show how interfacial concentration gradients are related to electric field strength and duration. Finally, we examine how discretization size affects the accuracy of these methods. Overall these methods are motivated by and well suited to addressing an outstanding goal: estimation of the net ionic and molecular transport facilitated by electroporation in biological systems.

  2. Rapid optimization of electroporation conditions for plant cells, protoplasts, and pollen.

    Science.gov (United States)

    Saunders, J A; Lin, C H; Hou, B H; Cheng, J; Tsengwa, N; Lin, J J; Smith, C R; McIntosh, M S; Van Wert, S

    1995-06-01

    The optimization of electroporation conditions for maximal uptake of DNA during direct gene transfer experiments is critical to achieve high levels of gene expression in transformed plant cells. Two stains, trypan blue and fluorescein diacetate, have been applied to optimize electroporation conditions for three plant cell types, using different square wave and exponential wave electroporation devices. The different cell types included protoplasts from tobacco, a stable mixotrophic suspension cell culture from soybean with intact cell walls, and germinating pollen from alfalfa and tobacco. Successful electroporation of each of these cell types was obtained, even in the presence of an intact cell wall when conditions were optimized for the electroporation pulse. The optimal field strength for each of these cells differs, protoplasts having the lowest optimal pulse field strength, followed by suspension cells and finally germinating pollen requiring the strongest electroporation pulse. A rapid procedure is described for optimizing electroporation parameters using different types of cells from different plant sources.

  3. Optimisation of Microstrip Antenna

    Directory of Open Access Journals (Sweden)

    H. El Hamchary

    1996-04-01

    Full Text Available When choosing the most appropriate microstrip antenna configuration for particular applications, the kind of excitation of the radiating element is an essential factor that requires careful considerations. For controlling the distribution of energy of the linear or planar array of elements and for coupling energy to the individual elements, a wide variety of feed mechanisms are available. In this paper, the coaxial antenna feeding is assumed and the best (optimised feeding is found. Then, antenna characteristics such as radiation pattern, return loss, input impedance, and VSWR are obtained.

  4. Calcium electroporation in three cell lines; a comparison of bleomycin and calcium, calcium compounds, and pulsing conditions

    DEFF Research Database (Denmark)

    Frandsen, Stine Krog; Gissel, Hanne; Hojman, Pernille

    2013-01-01

    BACKGROUND: Electroporation with calcium (calcium electroporation) can induce ATP depletion-associated cellular death. In the clinical setting, the cytotoxic drug bleomycin is currently used with electroporation (electrochemotherapy) for palliative treatment of tumors. Calcium electroporation...... offers several advantages over standard treatment options: calcium is inexpensive and may readily be applied without special precautions, as is the case with cytostatic drugs. Therefore, details on the use of calcium electroporation are essential for carrying out clinical trials comparing calcium...

  5. A Global Optimisation Toolbox for Massively Parallel Engineering Optimisation

    CERN Document Server

    Biscani, Francesco; Yam, Chit Hong

    2010-01-01

    A software platform for global optimisation, called PaGMO, has been developed within the Advanced Concepts Team (ACT) at the European Space Agency, and was recently released as an open-source project. PaGMO is built to tackle high-dimensional global optimisation problems, and it has been successfully used to find solutions to real-life engineering problems among which the preliminary design of interplanetary spacecraft trajectories - both chemical (including multiple flybys and deep-space maneuvers) and low-thrust (limited, at the moment, to single phase trajectories), the inverse design of nano-structured radiators and the design of non-reactive controllers for planetary rovers. Featuring an arsenal of global and local optimisation algorithms (including genetic algorithms, differential evolution, simulated annealing, particle swarm optimisation, compass search, improved harmony search, and various interfaces to libraries for local optimisation such as SNOPT, IPOPT, GSL and NLopt), PaGMO is at its core a C++ ...

  6. Electroporation: A New Approach Enhancing Antitumor Effects of Cytoxan

    Institute of Scientific and Technical Information of China (English)

    Yang Kong(杨孔); Yue Bisong; Wang Zishu; Zou Fangdong; Zhao Ermi; Wang Baoyi; Zhang Hong

    2003-01-01

    Electrochemotherapy (ECT) is a novel cancer treatment in which electric pulses (Eps) inducing cell membrane pored (electroporation) are used as a means of delivering antitumor drugs to the cytoplasm of cancer cells. In vitro, with scan electromicroscope (SEM) and Trypan blue staining examination, the best parameter of Eps of electroporation is studied by the S-180 cells exposed to EP with various voltages, pulses , capacitance. The best parameter of EP of electroporation is 600V/cm, 6 pulses, 10 μF. In the in vivo study, ECT is studied with the Cytoxan (CTX) injected directly into the tumor followed immediately by a local EP at the tumor site. Four parameters, which include the tumor inhibitory ratio, the curing ratio and the vas capillare of tumor, the tumor's histopathological characteristics are determined and compared among the ECT group, the control group, the EP-only group and the drug-only group. The results indicate that the antitumor effect of CTX is significantly enhanced by electroporation.

  7. Low vulnerability of the right phrenic nerve to electroporation ablation

    NARCIS (Netherlands)

    van Driel, Vincent J. H. M.; Neven, KGEJ; van Wessel, Harri; Vink, Aryan; Doevendans, Pieter A. F. M.; Wittkampf, Fred H. M.

    2015-01-01

    BACKGROUND Circular electroporation ablation is a novel ablation modality for electrical pulmonary vein isolation. With a single 200-3 application, deep circular myocardial lesions can be created. However, the acute and chronic effects of this energy source on phrenic nerve (PN) function are unknown

  8. Electroporation of cells in microfluidic devices: a review

    NARCIS (Netherlands)

    Fox, M.B.; Esveld, D.C.; Valero, A.; Luttge, R.; Mastwijk, H.C.; Bartels, P.V.; Berg, van den A.; Boom, R.M.

    2006-01-01

    In recent years, several publications on microfluidic devices have focused on the process of electroporation, which results in the poration of the biological cell membrane. The devices involved are designed for cell analysis, transfection or pasteurization. The high electric field strengths needed a

  9. D-glucosamine promotes transfection efficiency during electroporation.

    Science.gov (United States)

    Igawa, Kazunari; Ohara, Naoko; Kawakubo, Atsushi; Sugimoto, Kouji; Yanagiguchi, Kajiro; Ikeda, Takeshi; Yamada, Shizuka; Hayashi, Yoshihiko

    2014-01-01

    D-Glucosamine is a useful medicament in various fields of medicine and dentistry. With respect to stability of the cell membrane, it has been reported that bradykinin-induced nociceptive responses are significantly suppressed by the direct application of D-glucosamine. Electroporation is usually used to effectively introduce foreign genes into tissue culture cells. Buffers for electroporation with or without D-glucosamine are used in experiments of transfection vectors. This is the first study to indirectly observe the stability and protection of the osteoblast membrane against both electric stress and gene uptake (the proton sponge hypothesis: osmotic rupture during endosomes prior to fusion with lysosomes) in electroporation with D-glucosamine application. The transfection efficiency was evaluated as the fluorescence intensity of the transfected green fluorescent protein (GFP) in the cultured cells (osteoblasts; NOS-1 cells). The transfection efficiency increased over 30% in the electroporation samples treated with D-glucosamine-supplemented buffer after one day. The membrane absorption of D-glucosamine is the primary mechanism of membrane stress induced by electric stress. This new function of D-glucosamine is useful and meaningful for developing more effective transformation procedures.

  10. Transformation of undomesticated strains of Bacillus subtilis by protoplast electroporation

    NARCIS (Netherlands)

    Romero, Diego; Perez-Garcia, Alejandro; Veening, Jan-Willem; de Vicente, Antonio; Kuipers, Oscar P.; de, Vicente A.

    2006-01-01

    A rapid method combining the use of protoplasts and electroporation was developed to transform recalcitrant wild strains of Bacillus subtilis. The method described here allows transformation with both replicative and integrative plasmids, as well as with chromosomal DNA, and provides a valuable tool

  11. Detection of Kinase Translocation Using Microfluidic Electroporative Flow Cytometry

    Science.gov (United States)

    Lu, Chang; Wang, Jun; Bao, Ning; Paris, Leela; Wang, Hsiang-Yu; Geahlen, Robert

    2008-03-01

    Translocation of a protein between different subcellular compartments is a common event during signal transduction in living cells. Detection of these events has been largely carried out based on imaging of a low number of cells and subcellular fractionation/Western blotting. These conventional techniques either lack the high throughput desired for probing an entire cell population or provide only the average behaviors of cell populations without information from single cells. Here we demonstrate a new tool, referred to as microfluidic electroporative flow cytometry, to detect the translocation of an EGFP-tagged tyrosine kinase, Syk, to the plasma membrane in B cells at the level of the cell population. We combine electroporation with flow cytometry and observe the release of intracellular kinase out of the cells during electroporation. We found that the release of the kinase was strongly influenced by its subcellular localization. Cells stimulated through the antigen receptor have a fraction of the kinase at the plasma membrane and retain more kinase after electroporation than do cells without stimulation and translocation. This tool will have utility for kinase-related drug discovery and tumor diagnosis and staging.

  12. Transformation of undomesticated strains of Bacillus subtilis by protoplast electroporation.

    Science.gov (United States)

    Romero, Diego; Pérez-García, Alejandro; Veening, Jan-Willem; de Vicente, Antonio; Kuipers, Oscar P

    2006-09-01

    A rapid method combining the use of protoplasts and electroporation was developed to transform recalcitrant wild strains of Bacillus subtilis. The method described here allows transformation with both replicative and integrative plasmids, as well as with chromosomal DNA, and provides a valuable tool for molecular genetic analysis of interesting Bacillus strains, which are hard to transform by conventional methods.

  13. Regeneration of Transgenic Soybean (Glycine max) Plants from Electroporated Protoplasts.

    Science.gov (United States)

    Dhir, S K; Dhir, S; Savka, M A; Belanger, F; Kriz, A L; Farrand, S K; Widholm, J M

    1992-05-01

    Transgenic soybean (Glycine max [L.] Merr.) plants were regenerated from calli derived from protoplasts electroporated with plasmid DNA-carrying genes for a selectable marker, neomycin phosphotransferase (NPTII), under the control of the cauliflower mosaic virus 35-Svedberg unit promoter, linked with a nonselectable mannityl opine synthesis marker. Following electroporation and culture, the protoplast-derived colonies were subjected to kanamycin selection (50 micrograms per milliliter) beginning on day 15 for 6 weeks. Approximately, 370 to 460 resistant colonies were recovered from 1 x 10(6) electroporated protoplasts, giving an absolute transformation frequency of 3.7 to 4.6 x 10(-4). More than 80% of the kanamycin-resistant colonies showed NPTII activity, and about 90% of these also synthesized opines. This indicates that the linked marker genes were co-introduced and co-expressed at a very high frequency. Plants were regenerated from the transformed cell lines. Southern blot analysis of the transformed callus and leaf DNA demonstrated the integration of both genes. Single-plant assays performed with different plant parts showed that both shoot and root tissues express NPTII activity and accumulate opines. Experiments with NPTII and mannityl opine synthesis marker genes on separate plasmids resulted in a co-expression rate of 66%. These results indicate that electroporation can be used to introduce both linked and unlinked genes into the soybean to produce transformed plants.

  14. Transfection of E. coli with lambda DNA by electroporation.

    Science.gov (United States)

    Magistrelli, C; Colombo, E; Tognoni, A; Grandi, G

    1992-10-01

    In the ambit of the B. subtilis genoma sequencing and mapping project, we have set up an electroporation method to transfect E. coli cells with lambda DNA. This methodology presents features that make it preferable to traditional in vitro packaging for some purposes. Here we will illustrate the experimental procedure and the possible applications.

  15. Low vulnerability of the right phrenic nerve to electroporation ablation

    NARCIS (Netherlands)

    van Driel, Vincent J. H. M.; Neven, KGEJ; van Wessel, Harri; Vink, Aryan; Doevendans, Pieter A. F. M.|info:eu-repo/dai/nl/164248366; Wittkampf, Fred H. M.|info:eu-repo/dai/nl/080434436

    BACKGROUND Circular electroporation ablation is a novel ablation modality for electrical pulmonary vein isolation. With a single 200-3 application, deep circular myocardial lesions can be created. However, the acute and chronic effects of this energy source on phrenic nerve (PN) function are

  16. Gold nanoparticles electroporation enhanced polyplex delivery to mammalian cells.

    Science.gov (United States)

    Huang, Shuyan; Deshmukh, Harshavardhan; Rajagopalan, Kartik Kumar; Wang, Shengnian

    2014-07-01

    Nonviral methods have been explored as the replacement of viral systems for their low toxicity and immunogenicity. However, they have yet to reach levels competitive to their viral counterparts. In this paper, we combined physical and chemical methods to improve the performance of polyplex delivery of DNA and small interfering RNA. Specifically, gold nanoparticles (AuNPs) were used to carry polyplex (a chemical approach) while electroporation (a physical approach) was applied for fast and direct cytosolic delivery. In this hybrid approach, cationic polymer molecules condense and/or protect genetic probes as usual while AuNPs help fix polycations to reduce their cytotoxicity and promote the transfection efficiency of electroporation. AuNPs of various sizes were first coated with polyethylenimine, which were further conjugated with DNA plasmids or small interfering RNA molecules to form AuNPs-polyplex. The hybrid nanoparticles were then mixed with cells and introduced into cell cytosol by electroporation. The delivery efficiency was evaluated with both model anchor cells (i.e., NIH/3T3) and suspension cells (i.e., K562), together with their impact on cell viability. We found that AuNP-polyplex showed 1.5∼2 folds improvement on the transfection efficiency with no significant increase of toxicity when compared to free plasmid delivery by electroporation alone. Such a combination of physical and chemical delivery concept may stimulate further exploration in the delivery of various therapeutic materials for both in vitro and in vivo applications.

  17. Theoretical studies of lipid bilayer electroporation using molecular dynamics simulations

    Science.gov (United States)

    Levine, Zachary Alan

    Computer simulations of physical, chemical, and biological systems have improved tremendously over the past five decades. From simple studies of liquid argon in the 1960s to fully atomistic simulations of entire viruses in the past few years, recent advances in high-performance computing have continuously enabled simulations to bridge the gap between scientific theory and experiment. Molecular dynamics simulations in particular have allowed for the direct observation of spatial and temporal events which are at present inaccessible to experiments. For this dissertation I employ all-atom molecular dynamics simulations to study the transient, electric field-induced poration (or electroporation) of phospholipid bilayers at MV/m electric fields. Phospholipid bilayers are the dominant constituents of cell membranes and act as both a barrier and gatekeeper to the cell interior. This makes their structural integrity and susceptibility to external perturbations an important topic for study, especially as the density of electromagnetic radiation in our environment is increasing steadily. The primary goal of this dissertation is to understand the specific physical and biological mechanisms which facilitate electroporation, and to connect our simulated observations to experiments with live cells and to continuum models which seek to describe the underlying biological processes of electroporation. In Chapter 1 I begin with a brief introduction to phospholipids and phospholipid bilayers, followed by an extensive overview of electroporation and atomistic molecular dynamics simulations. The following chapters will then focus on peer-reviewed and published work we performed, or on existing projects which are currently being prepared for submission. Chapter 2 looks at how external electric fields affect both oxidized and unoxidized lipid bilayers as a function of oxidation concentration and oxidized lipid type. Oxidative damage to cell membranes represents a physiologically relevant

  18. Simple Combinatorial Optimisation Cost Games

    NARCIS (Netherlands)

    van Velzen, S.

    2005-01-01

    In this paper we introduce the class of simple combinatorial optimisation cost games, which are games associated to {0, 1}-matrices.A coalitional value of a combinatorial optimisation game is determined by solving an integer program associated with this matrix and the characteristic vector of the

  19. Determination of cell electroporation in small-volume samples.

    Science.gov (United States)

    Saulis, Gintautas; Praneviciŭte, Rita

    2007-01-01

    Expose of cells to electric field pulses increases the cell membrane permeability. Intracellular potassium ions leak out of the cells through aqueous pores created in the membrane. This release is used here for the determination of the fraction of electroporated cells. To determine cell membrane electroporation in small-volume samples (40-50 miacrol), mini both potassium ion-selective and reference electrodes, with tip diameters of 1-1.5 mm and minimum immersion depths of 1 mm, were utilized. The obtained calibration graph was linear within the concentration range 0.2-100 mM. The slope was 50-51 and 53-56 mV per concentration order at 10-11 and 19-21 degrees C, respectively. Detection limit of the electrode was determined to be 0.05-0.08 mM, however, it was possible to work down to concentrations in the range of 0.01 mM. Experiments have been carried out on human erythrocytes exposed to a square-wave electric pulse with the duration of 0.1-2 ms. The extracellular potassium concentrations were in the range between 0.04-0.08 mM (intact cells) and 3-5 mM (100% electroporation). The obtained dependences of the fraction of electroporated cells on the pulse intensity were of a sigmoid shape. The dependence of the pulse amplitude required to electroporate 50% of cells on the pulse duration, obtained from the release of intracellular potassium ions, coincided with the one determined from the extent of hemolysis after 24 h-incubation at low temperature.

  20. Electroporation of archaeal lipid membranes using MD simulations.

    Science.gov (United States)

    Polak, Andraž; Tarek, Mounir; Tomšič, Matija; Valant, Janez; Ulrih, Nataša Poklar; Jamnik, Andrej; Kramar, Peter; Miklavčič, Damijan

    2014-12-01

    Molecular dynamics (MD) simulations were used to investigate the electroporation of archaeal lipid bilayers when subjected to high transmembrane voltages induced by a charge imbalance, mimicking therefore millisecond electric pulse experiments. The structural characteristics of the bilayer, a 9:91 mol% 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-myo-inositol (AI) and 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-1'(2'-O-α-D-glucosyl)-myo-inositol (AGI) were compared to small angle X-ray scattering data. A rather good agreement of the electron density profiles at temperatures of 298 and 343 K was found assessing therefore the validity of the protocols and force fields used in simulations. Compared to dipalmitoyl-phosphatidylcholine (DPPC), the electroporation threshold for the bilayer was found to increase from ~2 V to 4.3 V at 323 K, and to 5.2 V at 298 K. Comparing the electroporation thresholds of the archaeal lipids to those of simple diphytanoyl-phosphatidylcholine (DPhPC) bilayers (2.5 V at 323 K) allowed one to trace back the stability of the membranes to the structure of their lipid head groups. Addition of DPPC in amounts of 50 mol% to the archaeal lipid bilayers decreases their stability and lowers the electroporation thresholds to 3.8 V and 4.1 V at respectively 323 and 298 K. The present study therefore shows how membrane compositions can be selected to cover a wide range of responses to electric stimuli. This provides new routes for the design of liposomes that can be efficiently used as drug delivery carriers, as the selection of their composition allows one to tune in their electroporation threshold for subsequent release of their load.

  1. Efficiency of cellular delivery of antisense peptide nucleic acid by electroporation depends on charge and electroporation geometry

    DEFF Research Database (Denmark)

    Joergensen, Mette; Agerholm-Larsen, Birgit; Nielsen, Peter E;

    2011-01-01

    Electroporation is potentially a very powerful technique for both in vitro cellular and in vivo drug delivery, particularly relating to oligonucleotides and their analogs for genetic therapy. Using a sensitive and quantitative HeLa cell luciferase RNA interference mRNA splice correction assay...

  2. The Effects of Irreversible Electroporation on the Achilles Tendon: An Experimental Study in a Rabbit Model.

    Directory of Open Access Journals (Sweden)

    Yue Song

    Full Text Available To evaluate the potential effects of irreversible electroporation ablation on the Achilles tendon in a rabbit model and to compare the histopathological and biomechanical changes between specimens following electroporation ablation and radiofrequency ablation.A total of 140 six-month-old male New Zealand rabbits were used. The animals were randomly divided into two groups, 70 in the radiofrequency ablation group and 70 in the electroporation group. In situ ablations were applied directly to the Achilles tendons of rabbits using typical electroporation (1800 V/cm, 90 pulses and radiofrequency ablation (power control mode protocols. Histopathological and biomechanical evaluations were performed to examine the effects of electroporation ablation and radiofrequency ablation over time.Both electroporation and radiofrequency ablation produced complete cell ablation in the target region. Thermal damage resulted in tendon rupture 3 days post radiofrequency ablation. In contrast, electroporation-ablated Achilles tendons preserved their biomechanical properties and showed no detectable rupture at this time point. The electroporation-ablated tendons exhibited signs of recovery, including tenoblast regeneration and angiogenesis within 2 weeks, and the restoration of their integral structure was evident within 12 weeks.When applying electroporation to ablate solid tumors, major advantage could be that collateral damage to adjacent tendons or ligaments is minimized due to the unique ability of electroporation ablation to target the cell membrane. This advantage could have a significant impact on the field of tumor ablation near vital tendons or ligaments.

  3. Topology optimised wavelength dependent splitters

    DEFF Research Database (Denmark)

    Hede, K. K.; Burgos Leon, J.; Frandsen, Lars Hagedorn

    A photonic crystal wavelength dependent splitter has been constructed by utilising topology optimisation1. The splitter has been fabricated in a silicon-on-insulator material (Fig. 1). The topology optimised wavelength dependent splitter demonstrates promising 3D FDTD simulation results....... This complex photonic crystal structure is very sensitive against small fabrication variations from the expected topology optimised design. A wavelength dependent splitter is an important basic building block for high-performance nanophotonic circuits. 1J. S. Jensen and O. Sigmund, App. Phys. Lett. 84, 2022...

  4. Engineering Optimisation by Cuckoo Search

    CERN Document Server

    Yang, Xin-She

    2010-01-01

    A new metaheuristic optimisation algorithm, called Cuckoo Search (CS), was developed recently by Yang and Deb (2009). This paper presents a more extensive comparison study using some standard test functions and newly designed stochastic test functions. We then apply the CS algorithm to solve engineering design optimisation problems, including the design of springs and welded beam structures. The optimal solutions obtained by CS are far better than the best solutions obtained by an efficient particle swarm optimiser. We will discuss the unique search features used in CS and the implications for further research.

  5. Topology optimised wavelength dependent splitters

    DEFF Research Database (Denmark)

    Hede, K. K.; Burgos Leon, J.; Frandsen, Lars Hagedorn;

    A photonic crystal wavelength dependent splitter has been constructed by utilising topology optimisation1. The splitter has been fabricated in a silicon-on-insulator material (Fig. 1). The topology optimised wavelength dependent splitter demonstrates promising 3D FDTD simulation results....... This complex photonic crystal structure is very sensitive against small fabrication variations from the expected topology optimised design. A wavelength dependent splitter is an important basic building block for high-performance nanophotonic circuits. 1J. S. Jensen and O. Sigmund, App. Phys. Lett. 84, 2022...

  6. In vivo electrical conductivity measurements during and after tumor electroporation: conductivity changes reflect the treatment outcome.

    Science.gov (United States)

    Ivorra, Antoni; Al-Sakere, Bassim; Rubinsky, Boris; Mir, Lluis M

    2009-10-01

    Electroporation is the phenomenon in which cell membrane permeability is increased by exposing the cell to short high-electric-field pulses. Reversible electroporation treatments are used in vivo for gene therapy and drug therapy while irreversible electroporation is used for tissue ablation. Tissue conductivity changes induced by electroporation could provide real-time feedback of the treatment outcome. Here we describe the results from a study in which fibrosarcomas (n = 39) inoculated in mice were treated according to different electroporation protocols, some of them known to cause irreversible damage. Conductivity was measured before, within the pulses, in between the pulses and for up to 30 min after treatment. Conductivity increased pulse after pulse. Depending on the applied electroporation protocol, the conductivity increase after treatment ranged from 10% to 180%. The most significant conclusion from this study is the fact that post-treatment conductivity seems to be correlated with treatment outcome in terms of reversibility.

  7. Development of an efficient electroporation method for rhizobacterial Bacillus mycoides strains.

    Science.gov (United States)

    Yi, Yanglei; Kuipers, Oscar P

    2017-02-01

    In order to develop a method for electroporation of environmental Bacillus mycoides strains, we optimized several conditions that affect the electroporation efficiency of this bacterium. By combining the optimized conditions, the electroporation efficiency of strain EC18 was improved to (1.3±0.6)×10(5)cfu/μg DNA, which is about 10(3)-fold increase in comparison with a previously reported value. The method was further validated on various B. mycoides strains, yielding reasonable transformation efficiencies. Furthermore, we confirmed that restriction/modification is the main barrier for electroporation of this bacterium. To the best of our knowledge, this is the first systematic investigation of various parameters of electroporation of B. mycoides. The electroporation method reported will allow for efficient genetic manipulation of this bacterium.

  8. Optimisation of the Laser Cutting Process

    DEFF Research Database (Denmark)

    Dragsted, Birgitte; Olsen, Flemmming Ove

    1996-01-01

    The problem in optimising the laser cutting process is outlined. Basic optimisation criteria and principles for adapting an optimisation method, the simplex method, are presented. The results of implementing a response function in the optimisation are discussed with respect to the quality as well...

  9. Macroscopic characterization of cell electroporation in biological tissue based on electrical measurements

    Science.gov (United States)

    Cima, Lionel F.; Mir, Lluis M.

    2004-11-01

    A method is described to experimentally determine the temporal evolution of state variables involved in the electroporation of biological tissue, i.e., the transmembrane voltage and the macroscopic current flowing in the electropores. Indeed, the electrical parameters of the extracellular, intracellular, and unaltered membrane contributions as well as the electropores electrical characteristics can be deduced from the measurement of the tissue bioimpedance and from the variations of both the macroscopic voltage applied to the tissue and the delivered current.

  10. Selective gene expression by postnatal electroporation during olfactory interneuron neurogenesis.

    Directory of Open Access Journals (Sweden)

    Alexander T Chesler

    Full Text Available Neurogenesis persists in the olfactory system throughout life. The mechanisms of how new neurons are generated, how they integrate into circuits, and their role in coding remain mysteries. Here we report a technique that will greatly facilitate research into these questions. We found that electroporation can be used to robustly and selectively label progenitors in the Subventicular Zone. The approach was performed postnatally, without surgery, and with near 100% success rates. Labeling was found in all classes of interneurons in the olfactory bulb, persisted to adulthood and had no adverse effects. The broad utility of electroporation was demonstrated by encoding a calcium sensor and markers of intracellular organelles. The approach was found to be effective in wildtype and transgenic mice as well as rats. Given its versatility, robustness, and both time and cost effectiveness, this method offers a powerful new way to use genetic manipulation to understand adult neurogenesis.

  11. Microscale Symmetrical Electroporator Array as a Versatile Molecular Delivery System

    Science.gov (United States)

    Ouyang, Mengxing; Hill, Winfield; Lee, Jung Hyun; Hur, Soojung Claire

    2017-03-01

    Successful developments of new therapeutic strategies often rely on the ability to deliver exogenous molecules into cytosol. We have developed a versatile on-chip vortex-assisted electroporation system, engineered to conduct sequential intracellular delivery of multiple molecules into various cell types at low voltage in a dosage-controlled manner. Micro-patterned planar electrodes permit substantial reduction in operational voltages and seamless integration with an existing microfluidic technology. Equipped with real-time process visualization functionality, the system enables on-chip optimization of electroporation parameters for cells with varying properties. Moreover, the system’s dosage control and multi-molecular delivery capabilities facilitate intracellular delivery of various molecules as a single agent or in combination and its utility in biological research has been demonstrated by conducting RNA interference assays. We envision the system to be a powerful tool, aiding a wide range of applications, requiring single-cell level co-administrations of multiple molecules with controlled dosages.

  12. A new equivalent circuit model for micro electroporation systems

    KAUST Repository

    Shagoshtasbi, Hooman

    2011-02-01

    Electroporation (EP) is a unique biotechnique in which intense electric pulses are applied on the cell membrane to temporarily generate nanoscale electropores and to increase the membrane permeability for the delivery of exogenous biomolecules or drugs. We propose a new equivalent circuit model with 8 electric components to predict the electrodynamic response of a micro EP system. As the permeability of the cell membrane increases, the membrane resistance decreases. The numerical simulations of the transmembrane current responses to different applied voltages (1∼6V) are consistent with the experimental results using HeLa cells. Besides, the transmembrane voltage as a function of applied voltages is determined as well. These transmembrane current and voltage responses can be extremely useful for the design of new generation of micro EP systems for transfection of large DNA molecules in the future. © 2011 IEEE.

  13. Normal and Malignant Cells Exhibit Differential Responses to Calcium Electroporation

    DEFF Research Database (Denmark)

    Frandsen, Stine K; Krüger, Mie B; Mangalanathan, Uma M

    2017-01-01

    tissue after calcium electroporation but decreased in skin tissue 4 hours after treatment to levels comparable with untreated controls, whereas calcium content endured at high levels in tumor tissue. Mechanistic experiments in vitro indicated that calcium influx was similar in fibroblasts and cancer...... necrosis, with a range of sensitivities observed (36%-88%) 2 days after treatment. Necrosis was induced using calcium concentrations of 100-500 mmol/L and injection volumes 20%-80% of tumor volume. Notably, only limited effects were seen in normal tissue. Calcium content increased >7-fold in tumor and skin......Calcium electroporation may offer a simple general tool for anticancer therapy. Transient permeabilization of cancer cell membranes created by applying short, high-voltage pulses in tumors enables high calcium influxes that trigger cell death. In this study, we compared the relative sensitivity...

  14. Isogeometric Analysis and Shape Optimisation

    DEFF Research Database (Denmark)

    Gravesen, Jens; Evgrafov, Anton; Gersborg, Allan Roulund

    obtained and also some of the problems we have encountered. One of these problems is that the geometry of the shape is given by the boundary alone. And, it is the parametrisation of the boundary which is changed by the optimisation procedure. But isogeometric analysis requires a parametrisation......One of the attractive features of isogeometric analysis is the exact representation of the geometry. The geometry is furthermore given by a relative low number of control points and this makes isogeometric analysis an ideal basis for shape optimisation. I will describe some of the results we have...... of the whole domain. So in every optimisation cycle we need to extend a parametrisation of the boundary of a domain to the whole domain. It has to be fast in order not to slow the optimisation down but it also has to be robust and give a parametrisation of high quality. These are conflicting requirements so we...

  15. Turbulence optimisation in stellarator experiments

    Energy Technology Data Exchange (ETDEWEB)

    Proll, Josefine H.E. [Max-Planck/Princeton Center for Plasma Physics (Germany); Max-Planck-Institut fuer Plasmaphysik, Wendelsteinstr. 1, 17491 Greifswald (Germany); Faber, Benjamin J. [HSX Plasma Laboratory, University of Wisconsin-Madison, Madison, WI 53706 (United States); Helander, Per; Xanthopoulos, Pavlos [Max-Planck/Princeton Center for Plasma Physics (Germany); Lazerson, Samuel A.; Mynick, Harry E. [Plasma Physics Laboratory, Princeton University, P.O. Box 451 Princeton, New Jersey 08543-0451 (United States)

    2015-05-01

    Stellarators, the twisted siblings of the axisymmetric fusion experiments called tokamaks, have historically suffered from confining the heat of the plasma insufficiently compared with tokamaks and were therefore considered to be less promising candidates for a fusion reactor. This has changed, however, with the advent of stellarators in which the laminar transport is reduced to levels below that of tokamaks by shaping the magnetic field accordingly. As in tokamaks, the turbulent transport remains as the now dominant transport channel. Recent analytical theory suggests that the large configuration space of stellarators allows for an additional optimisation of the magnetic field to also reduce the turbulent transport. In this talk, the idea behind the turbulence optimisation is explained. We also present how an optimised equilibrium is obtained and how it might differ from the equilibrium field of an already existing device, and we compare experimental turbulence measurements in different configurations of the HSX stellarator in order to test the optimisation procedure.

  16. Transformation of undomesticated strains of Bacillus subtilis by protoplast electroporation

    OpenAIRE

    Romero, Diego; Perez-Garcia, Alejandro; Veening, Jan-Willem; Vicente, Antonio; Oscar P. Kuipers; Vicente A.

    2006-01-01

    A rapid method combining the use of protoplasts and electroporation was developed to transform recalcitrant wild strains of Bacillus subtilis. The method described here allows transformation with both replicative and integrative plasmids, as well as with chromosomal DNA, and provides a valuable tool for molecular genetic analysis of interesting Bacillus strains, which are hard to transform by conventional methods. (c) 2006 Elsevier B.V. All rights reserved.

  17. The dissection of meiotic chromosome movement in mice using an in vivo electroporation technique

    National Research Council Canada - National Science Library

    Shibuya, Hiroki; Morimoto, Akihiro; Watanabe, Yoshinori

    2014-01-01

    .... Here, applying an in vivo electroporation (EP) technique in live mouse testis, we achieved the quick visualization of telomere, chromosome axis and microtubule organizing center (MTOC) movements...

  18. Electroporation loading of membrane-impermeable molecules to investigate intra- and intercellular Ca2+ signaling.

    Science.gov (United States)

    Decrock, Elke; De Bock, Marijke; Wang, Nan; Bol, Mélissa; Gadicherla, Ashish K; Leybaert, Luc

    2015-03-02

    Electroporation is a technique that temporarily induces pores in the plasma membranes of cells, thereby allowing plasma membrane-impermeable substances to enter the cells. This loading method requires an electrical drive circuit providing an electroporation signal, an electrode to apply the signal to a localized zone in a cell monolayer, and a special solution that has a low electrical conductivity. To avoid impairment of cell function and cell death from the electroporation procedure itself, the applied electrical signal should ideally be a high-frequency oscillating signal (50 kHz) without any direct current (DC) component. Here, we describe the detailed procedure of electroporation loading.

  19. Design, modelling and preliminary characterisation of microneedle-based electrodes for tissue electroporation in vivo

    Science.gov (United States)

    O'Mahony, Conor; Houlihan, Ruth; Grygoryev, Konstantin; Ning, Zhenfei; Williams, John; Moore, Tom

    2016-10-01

    We analysed the use of microneedle-based electrodes to enhance electroporation of mouse testis with DNA vectors for production of transgenic mice. Different microneedle formats were developed and tested, and we ultimately used electrodes based on arrays of 500 μm tall microneedles. In a series of experiments involving injection of a DNA vector expressing Green Fluorescent Protein (GFP) and electroporation using microneedle electrodes and a commercially available voltage supply, we compared the performance of flat and microneedle electrodes by measuring GFP expression at various timepoints after electroporation. Our main finding, supported by both experimental and simulated data, is that needles significantly enhanced electroporation of testis.

  20. Modeling of Transmembrane Potential in Realistic Multicellular Structures before Electroporation.

    Science.gov (United States)

    Murovec, Tomo; Sweeney, Daniel C; Latouche, Eduardo; Davalos, Rafael V; Brosseau, Christian

    2016-11-15

    Many approaches for studying the transmembrane potential (TMP) induced during the treatment of biological cells with pulsed electric fields have been reported. From the simple analytical models to more complex numerical models requiring significant computational resources, a gamut of methods have been used to recapitulate multicellular environments in silico. Cells have been modeled as simple shapes in two dimensions as well as more complex geometries attempting to replicate realistic cell shapes. In this study, we describe a method for extracting realistic cell morphologies from fluorescence microscopy images to generate the piecewise continuous mesh used to develop a finite element model in two dimensions. The preelectroporation TMP induced in tightly packed cells is analyzed for two sets of pulse parameters inspired by clinical irreversible electroporation treatments. We show that high-frequency bipolar pulse trains are better, and more homogeneously raise the TMP of tightly packed cells to a simulated electroporation threshold than conventional irreversible electroporation pulse trains, at the expense of larger applied potentials. Our results demonstrate the viability of our method and emphasize the importance of considering multicellular effects in the numerical models used for studying the response of biological tissues exposed to electric fields. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  1. Irreversible Electroporation for Focal Ablation at the Porta Hepatis

    Energy Technology Data Exchange (ETDEWEB)

    Kasivisvanathan, Veeru, E-mail: vk103@ic.ac.uk [Imperial College London, Department of Radiology (United Kingdom); Thapar, Ankur, E-mail: a.thapar09@imperial.ac.uk; Oskrochi, Youssof, E-mail: Youssof.Oskrochi09@imperial.ac.uk [Imperial College London, Department of Surgery and Cancer (United Kingdom); Picard, John, E-mail: John.picard@imperial.nhs.uk [Imperial College Healthcare NHS Trust, Department of Anaesthesia (United Kingdom); Leen, Edward L. S., E-mail: Edward.leen@imperial.ac.uk [Imperial College London, Department of Radiology (United Kingdom)

    2012-12-15

    Patients with chemotherapy-refractory liver metastases who are not candidates for surgery may be treated with focal ablation techniques with established survival benefits. Irreversible electroporation is the newest of these and has the putative advantages of a nonthermal action, preventing damage to adjacent biliary structures and bowel. This report describes the use of irreversible electroporation in a 61-year-old man with a solitary chemoresistant liver metastasis unsuitable for radiofrequency ablation as a result of its proximity to the porta hepatis. At 3 months, tumor size was decreased on computed tomography from 28 Multiplication-Sign 19 to 20 Multiplication-Sign 17 mm, representing stable disease according to the response evaluation criteria in solid tumors. This corresponded to a decrease in tumor volume size from 5.25 to 3.16 cm{sup 3}. There were no early or late complications. Chemoresistant liver metastases in the proximity of the porta hepatis that are considered to be too high a risk for conventional surgery or thermal ablation may be considered for treatment by the novel ablation technique of irreversible electroporation.

  2. A propagating heat wave model of skin electroporation.

    Science.gov (United States)

    Pliquett, Uwe; Gusbeth, Ch; Nuccitelli, Richard

    2008-03-21

    The main barrier to transdermal drug delivery in human skin is the stratum corneum. Pulsed electric fields (PEFs) of sufficient amplitude can create new aqueous pathways across this barrier and enhance drug delivery through the skin. Here, we describe a model of pore formation between adjacent corneocytes that predicts the following sequence of events: (1) the PEF rapidly charges the stratum corneum near the electrode until the transepidermal potential difference is large enough to drive water into a small region of the stratum corneum, creating new aqueous pathways. (2) PEFs then drive a high current density through this newly created electropore to generate Joule heating that warms the pore perimeter. (3) This temperature rise at the perimeter increases the probability of further electroporation there as the local sphingolipids reach their phase transition temperature. (4) This heat-generated wave of further electroporation propagates outward until the surface area of the pore becomes so large that the reduced current density no longer generates sufficient heat to reach the phase transition temperature of the sphingolipids. (5) Cooling and partial recovery occurs after the field pulse. This process yields large, high permeability regions in the stratum corneum at which molecules can more readily cross this skin barrier. We present a model for this process that predicts that the initial radius of the first aqueous pathway is approximately 5nm for a transdermal voltage of 60V at room temperature.

  3. DNA vaccines, electroporation and their applications in cancer treatment.

    Science.gov (United States)

    Lee, Si-Hyeong; Danishmalik, Sayyed Nilofar; Sin, Jeong-Im

    2015-01-01

    Numerous animal studies and recent clinical studies have shown that electroporation-delivered DNA vaccines can elicit robust Ag-specific CTL responses and reduce disease severity. However, cancer antigens are generally poorly immunogenic, requiring special conditions for immune response induction. To date, many different approaches have been used to elicit Ag-specific CTL and anti-neoplastic responses to DNA vaccines against cancer. In vivo electroporation is one example, whereas others include DNA manipulation, xenogeneic antigen use, immune stimulatory molecule and immune response regulator application, DNA prime-boost immunization strategy use and different DNA delivery methods. These strategies likely increase the immunogenicity of cancer DNA vaccines, thereby contributing to cancer eradication. However, cancer cells are heterogeneous and might become CTL-resistant. Thus, understanding the CTL resistance mechanism(s) employed by cancer cells is critical to develop counter-measures for this immune escape. In this review, the use of electroporation as a DNA delivery method, the strategies used to enhance the immune responses, the cancer antigens that have been tested, and the escape mechanism(s) used by tumor cells are discussed, with a focus on the progress of clinical trials using cancer DNA vaccines.

  4. Optimisation of load control

    Energy Technology Data Exchange (ETDEWEB)

    Koponen, P. [VTT Energy, Espoo (Finland)

    1998-08-01

    Electricity cannot be stored in large quantities. That is why the electricity supply and consumption are always almost equal in large power supply systems. If this balance were disturbed beyond stability, the system or a part of it would collapse until a new stable equilibrium is reached. The balance between supply and consumption is mainly maintained by controlling the power production, but also the electricity consumption or, in other words, the load is controlled. Controlling the load of the power supply system is important, if easily controllable power production capacity is limited. Temporary shortage of capacity causes high peaks in the energy price in the electricity market. Load control either reduces the electricity consumption during peak consumption and peak price or moves electricity consumption to some other time. The project Optimisation of Load Control is a part of the EDISON research program for distribution automation. The following areas were studied: Optimization of space heating and ventilation, when electricity price is time variable, load control model in power purchase optimization, optimization of direct load control sequences, interaction between load control optimization and power purchase optimization, literature on load control, optimization methods and field tests and response models of direct load control and the effects of the electricity market deregulation on load control. An overview of the main results is given in this chapter

  5. Electroporation of DC-3F cells is a dual process.

    Science.gov (United States)

    Wegner, Lars H; Frey, Wolfgang; Silve, Aude

    2015-04-01

    Treatment of biological material by pulsed electric fields is a versatile technique in biotechnology and biomedicine used, for example, in delivering DNA into cells (transfection), ablation of tumors, and food processing. Field exposure is associated with a membrane permeability increase usually ascribed to electroporation, i.e., formation of aqueous membrane pores. Knowledge of the underlying processes at the membrane level is predominantly built on theoretical considerations and molecular dynamics (MD) simulations. However, experimental data needed to monitor these processes with sufficient temporal resolution are scarce. The whole-cell patch-clamp technique was employed to investigate the effect of millisecond pulsed electric fields on DC-3F cells. Cellular membrane permeabilization was monitored by a conductance increase. For the first time, to our knowledge, it could be established experimentally that electroporation consists of two clearly separate processes: a rapid membrane poration (transient electroporation) that occurs while the membrane is depolarized or hyperpolarized to voltages beyond so-called threshold potentials (here, +201 mV and -231 mV, respectively) and is reversible within ∼100 ms after the pulse, and a long-term, or persistent, permeabilization covering the whole voltage range. The latter prevailed after the pulse for at least 40 min, the postpulse time span tested experimentally. With mildly depolarizing or hyperpolarizing pulses just above threshold potentials, the two processes could be separated, since persistent (but not transient) permeabilization required repetitive pulse exposure. Conductance increased stepwise and gradually with depolarizing and hyperpolarizing pulses, respectively. Persistent permeabilization could also be elicited by single depolarizing/hyperpolarizing pulses of very high field strength. Experimental measurements of propidium iodide uptake provided evidence of a real membrane phenomenon, rather than a mere

  6. Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation

    Directory of Open Access Journals (Sweden)

    J. Michael eGee

    2015-04-01

    Full Text Available Complex interactions between networks of astrocytes and neurons are beginning to be appreciated, but remain poorly understood. Transgenic mice expressing fluorescent protein reporters of cellular activity, such as the GCaMP family of genetically encoded calcium indicators, have been used to explore network behavior. However, in some cases, it may be desirable to use long-established rat models that closely mimic particular aspects of human conditions such as Parkinson’s disease and the development of epilepsy following status epilepticus. Methods for expressing reporter proteins in the rat brain are relatively limited. Transgenic rat technologies exist but are fairly immature. Viral-mediated expression is robust but unstable, requires invasive injections, and only works well for fairly small genes (< 5 kb. In utero electroporation offers a valuable alternative. IUE is a proven method for transfecting populations of astrocytes and neurons in the rat brain without the strict limitations on transgene size. We built a toolset of IUE plasmids carrying GCaMP variants 3, 6s or 6f driven by CAG and targeted to the cytosol or the plasma membrane. Because low baseline fluorescence of GCaMP can hinder identification of transfected cells, we included the option of co-expressing a cytosolic tdTomato protein. A binary system consisting of a plasmid carrying a piggyBac inverted terminal repeat-flanked CAG-GCaMP-IRES-tdTomato cassette and a separate plasmid encoding for expression of piggyBac transposase was employed to stably express GCaMP and tdTomato. The plasmids were co-electroporated on embryonic days 13.5-14.5 and astrocytic and neuronal activity was subsequently imaged in acute or cultured brain slices prepared from the cortex or hippocampus. Large spontaneous transients were detected in slices obtained from rats of varying ages up to 127 days. In this report, we demonstrate the utility of this toolset for interrogating astrocytic and neuronal

  7. Effects of electroporation on primary rat hepatocytes in vitro

    Institute of Scientific and Technical Information of China (English)

    Yun-Qing Yao; Ding-Feng Zhang; Ai-Long Huang; Yun Luo; Da-Zhi Zhang; Bo Wang; Wei-Ping Zhou; Hong Ren; Shu-Hua Guo

    2002-01-01

    AIM: To investigate the effects of electroporation on primaryrat hepatocyte and to optimize the electroporation conditionsintroducing foreign genes into primary hepatocytes.METHODS: A single-pulse procedure was performed at Iowvoltage (220-400 V) but with high capacitance (500-950 μF).Hepatocytes were divided into 4 groups according to theelectroporation conditions: group Ⅰ, 220 V and 500 μF;group Ⅱ, 220 Vand 950 μF; group Ⅲ, 400 V and 950 μF,and group Ⅳ.The control group was freshly isolatedhepatocytes and directly cultured under the same conditionsas those of electroporation groups. The effects ofelectroporation on primary rat hepatocytes were detectedby trypan blue exclusion (TBE) and MTT analysis. Besides,albumin (AIb), alanine transaminase (ALT) and lactatedehydrogenase (LDH) in the supernatants of culturedhepatocytes were measured by biochemical assay.RESULTS: Between day 1 and day 15 after incubation,primary rat hepatocytes of each electroporation groupappeared normal, being the same with those of controlgroup. TBE staining showed that slight hepatocyte damageand high survival rate were found in the electroporationgroups and the control group. Cultured for 3, 7, 11 and 15days, hepatocyte viability was approximatly 92.6±2.5 %,89.5±3.3 %, 82.0±3.5 % and 74.3±1.2 %, respectively.MTT analysis indicated that the viabilities of hepatocyteshad no significant difference between each electroporationgroup, and those were similar to that of control group. Atthe 36th hour after electroporation, AIb, ALT and LDH in thesupernatants of control group were 5.3±0.1 g. L-1, 183.7±8.4 nkat. L-1 and 896.8±58.5 nkat. L-1; those of group Ⅱwere 5.7±0.1 g. L-1, 215.4±16.7 nkat. L-1 and 1063.8±51.8 nkat. L-1; and those of group Ⅲ were 5.80.2 g. L-1,217.1 ± 8.4 nkat. L-1 and 1063.8± 10.0 nkat. L-1 . Statistically,the proteins of group Ⅱ and group Ⅲ were significantlyhigher than those of control group (P<0.05), whereas theprotein production of group

  8. Transfection of small numbers of human endothelial cells by electroporation and synthetic amphiphiles

    NARCIS (Netherlands)

    van Leeuwen, E B; van der Veen, A Y; Hoekstra, D; Engberts, J B; Halie, M R; van der Meer, J; Ruiters, M H

    1999-01-01

    OBJECTIVES: This study compared the efficiency of electroporation and synthetic amphiphiles. (SAINT-2pp/DOPE) in transfecting small numbers of human endothelial cells. METHODS AND RESULTS: Optimal transfection conditions were tested and appeared to be 400 V and 960 microF for electroporation and a 1

  9. The Influence of Soft Layer Electrokinetics on Electroporation of Gram-positive Bacteria

    Science.gov (United States)

    Dingari, Naga Neehar; Moran, Jeffrey L.; Garcia, Paulo A.; Buie, Cullen R.

    2016-11-01

    Bacterial electroporation involves subjecting cells to intense ( 10 kV/cm) electric pulses, to open pores on the cell membrane for intracellular delivery of exogenous molecules. Its high efficiency in genetic transformation makes it an attractive tool for synthetic biology. While mammalian cell electroporation has received extensive theoretical and experimental investigation, bacterial electroporation has received markedly less attention. In this work, we develop a theoretical model of electroporation for gram-positive bacteria, taking into account the effect of the bacterial cell envelope on the cell's response to an electroporation pulse. We model the influence of the cell wall charge on the electrokinetic transport (and hence the pore properties) around the bacterial cell envelope using the Poisson-Nernst-Planck equations. Further, we account for the influence of the cell wall's mechanical elasticity on the pore radius evolution during electroporation, which is typically neglected in mammalian cell electroporation. This yields valuable information about favorable conditions for pore formation and will enable designing optimal platforms for bacteria electroporation.

  10. Development of an efficient electroporation method for iturin A-producing Bacillus subtilis ZK.

    Science.gov (United States)

    Zhang, Zhi; Ding, Zhong-Tao; Shu, Dan; Luo, Di; Tan, Hong

    2015-04-01

    In order to efficiently introduce DNA into B. subtilis ZK, which produces iturin A at a high level, we optimized seven electroporation conditions and explored an efficient electroporation method. Using the optimal conditions, the electroporation efficiency was improved to 1.03 × 10(70 transformants/μg of DNA, an approximately 10,000-fold increase in electroporation efficiency. This efficiency is the highest electroporation efficiency for B. subtilis and enables the construction of a directed evolution library or the knockout of a gene in B. subtilis ZK for molecular genetics studies. In the optimization process, the combined effects of three types of wall-weakening agents were evaluated using a response surface methodology (RSM) design, which led to a two orders of magnitude increase in electroporation efficiency. To the best of our limited knowledge, this study provides the first demonstration of using an RSM design for optimization of the electroporation conditions for B. subtilis. To validate the electroporation efficiency, a case study was performed and a gene (rapC) was inactivated in B. subtilis ZK using a suicide plasmid pMUTIN4. Moreover, we found that the rapC mutants exhibited a marked decrease in iturin A production, suggesting that the rapC gene was closely related to the iturin A production.

  11. TEM turbulence optimisation in stellarators

    CERN Document Server

    Proll, J H E; Xanthopoulos, P; Lazerson, S A; Faber, B J

    2015-01-01

    With the advent of neoclassically optimised stellarators, optimising stellarators for turbulent transport is an important next step. The reduction of ion-temperature-gradient-driven turbulence has been achieved via shaping of the magnetic field, and the reduction of trapped-electron mode (TEM) turbulence is adressed in the present paper. Recent analytical and numerical findings suggest TEMs are stabilised when a large fraction of trapped particles experiences favourable bounce-averaged curvature. This is the case for example in Wendelstein 7-X [C.D. Beidler $\\textit{et al}$ Fusion Technology $\\bf{17}$, 148 (1990)] and other Helias-type stellarators. Using this knowledge, a proxy function was designed to estimate the TEM dynamics, allowing optimal configurations for TEM stability to be determined with the STELLOPT [D.A. Spong $\\textit{et al}$ Nucl. Fusion $\\bf{41}$, 711 (2001)] code without extensive turbulence simulations. A first proof-of-principle optimised equilibrium stemming from the TEM-dominated stella...

  12. Direct therapeutic applications of calcium electroporation to effectively induce tumor necrosis

    DEFF Research Database (Denmark)

    Frandsen, Stine Krog; Gissel, Hanne; Hojman, Pernille;

    2012-01-01

    Electroporation of cells with short, high-voltage pulses causes a transient permeabilization of cell membranes that permits passage of otherwise nonpermeating ions and molecules. In this study, we illustrate how electroporation with isotonic calcium can achieve highly effective cancer cell kill...... in vivo. Calcium electroporation elicited dramatic antitumor responses in which 89% of treated tumors were eliminated. Histologic analyses indicated complete tumor necrosis. Mechanistically, calcium electroporation caused acute ATP depletion likely due to a combination of increased cellular use of ATP......, decreased production of ATP due to effects on the mitochondria, as well as loss of ATP through the permeabilized cell membrane. Taken together, our findings offer a preclinical proof of concept for the use of electroporation to load cancer cells with calcium as an efficient anticancer treatment...

  13. A detailed description of an economical setup for electroporation of chick embryos in ovo.

    Science.gov (United States)

    Borges, R M; Horne, J H; Melo, A; Vidal, J T; Vieceli, F M; Melo, M O; Kanno, T Y N; Fraser, S E; Yan, C Y I

    2013-09-01

    One of the challenges of the postgenomic era is characterizing the function and regulation of specific genes. For various reasons, the early chick embryo can easily be adopted as an in vivo assay of gene function and regulation. The embryos are robust, accessible, easily manipulated, and maintained in the laboratory. Genomic resources centered on vertebrate organisms increase daily. As a consequence of optimization of gene transfer protocols by electroporation, the chick embryo will probably become increasingly popular for reverse genetic analysis. The challenge of establishing chick embryonic electroporation might seem insurmountable to those who are unfamiliar with experimental embryological methods. To minimize the cost, time, and effort required to establish a chick electroporation assay method, we describe and illustrate in great detail the procedures involved in building a low-cost electroporation setup and the basic steps of electroporation.

  14. Nonlinear electro-mechanobiological behavior of cell membrane during electroporation

    KAUST Repository

    Deng, Peigang

    2012-01-01

    A nonlinear electroporation (EP) model is proposed to study the electro-mechanobiological behavior of cell membrane during EP, by taking the nonlinear large deformation of the membrane into account. The proposed model predicts the critical transmembrane potential and the activation energy for EP, the equilibrium pore size, and the resealing process of the pore. Single-cell EP experiments using a micro EP chip were conducted on chicken red blood cells at different temperatures to determine the activation energy and the critical transmembrane potential for EP. The experimental results are in good agreement with the theoretical predictions. © 2012 American Institute of Physics.

  15. Mediatization

    DEFF Research Database (Denmark)

    Hjarvard, Stig

    2017-01-01

    Mediatization research shares media effects studies' ambition of answering the difficult questions with regard to whether and how media matter and influence contemporary culture and society. The two approaches nevertheless differ fundamentally in that mediatization research seeks answers...... to these general questions by distinguishing between two concepts: mediation and mediatization. The media effects tradition generally considers the effects of the media to be a result of individuals being exposed to media content, i.e. effects are seen as an outcome of mediated communication. Mediatization...... research is concerned with long-term structural changes involving media, culture, and society, i.e. the influences of the media are understood in relation to how media are implicated in social and cultural changes and how these processes come to create new conditions for human communication and interaction...

  16. Cogeneration technologies, optimisation and implementation

    CERN Document Server

    Frangopoulos, Christos A

    2017-01-01

    Cogeneration refers to the use of a power station to deliver two or more useful forms of energy, for example, to generate electricity and heat at the same time. This book provides an integrated treatment of cogeneration, including a tour of the available technologies and their features, and how these systems can be analysed and optimised.

  17. Optimisation of solar synoptic observations

    Science.gov (United States)

    Klvaña, Miroslav; Sobotka, Michal; Švanda, Michal

    2012-09-01

    The development of instrumental and computer technologies is connected with steadily increasing needs for archiving of large data volumes. The current trend to meet this requirement includes the data compression and growth of storage capacities. This approach, however, has technical and practical limits. A further reduction of the archived data volume can be achieved by means of an optimisation of the archiving that consists in data selection without losing the useful information. We describe a method of optimised archiving of solar images, based on the selection of images that contain a new information. The new information content is evaluated by means of the analysis of changes detected in the images. We present characteristics of different kinds of image changes and divide them into fictitious changes with a disturbing effect and real changes that provide a new information. In block diagrams describing the selection and archiving, we demonstrate the influence of clouds, the recording of images during an active event on the Sun, including a period before the event onset, and the archiving of long-term history of solar activity. The described optimisation technique is not suitable for helioseismology, because it does not conserve the uniform time step in the archived sequence and removes the information about solar oscillations. In case of long-term synoptic observations, the optimised archiving can save a large amount of storage capacities. The actual capacity saving will depend on the setting of the change-detection sensitivity and on the capability to exclude the fictitious changes.

  18. For Time-Continuous Optimisation

    DEFF Research Database (Denmark)

    Heinrich, Mary Katherine; Ayres, Phil

    2016-01-01

    Strategies for optimisation in design normatively assume an artefact end-point, disallowing continuous architecture that engages living systems, dynamic behaviour, and complex systems. In our Flora Robotica investigations of symbiotic plant-robot bio-hybrids, we re- quire computational tools...

  19. For Time-Continuous Optimisation

    DEFF Research Database (Denmark)

    Heinrich, Mary Katherine; Ayres, Phil

    2016-01-01

    Strategies for optimisation in design normatively assume an artefact end-point, disallowing continuous architecture that engages living systems, dynamic behaviour, and complex systems. In our Flora Robotica investigations of symbiotic plant-robot bio-hybrids, we re- quire computational tools...

  20. Influence of electroporation on chicken blastoderm cell viability in vitro.

    Science.gov (United States)

    Wawrzynska, Magdalena; Bednarczyk, Marek; Łakota, Pawel; Lubiszewska, Marta

    2008-01-01

    The aim of this study was to compare two types of devices used for blastoderm cell (BC) transfection: the Nucleofector (Amaxa, Biosystems) and the Multiporator (Eppendorf). To assess the influence of electric current on BCs, different conditions of both nucleofection and electroporation were used. Next, the viability of cells was assessed. The highest number of cells (90.8%) was viable after nucleofection in the G10 program. After transfection in the presence of pmaxGFP, the A23 program was found to be most advantageous. The elecroporation experiment with the Multiporator (Eppendorf) showed a significant influence of osmotic pressure and voltage on BC viability. Namely, in the isoosmolar buffer BC viability was statistically higher (P < or = 0.05) in comparison to the hypoosmolar buffer. The, viability of cells was statistically higher (P < or = 0.05) after application of 25V as compared to 50V. The efficiency of transfection in the presence of EGFP-C 1 after electroporation in 2 pulses, 25V, 500 micros in the isoosmolar buffer was better than in the recommended conditions in the Amaxa Biosystems A23 program.

  1. The role of pH fronts in reversible electroporation.

    Directory of Open Access Journals (Sweden)

    Pablo Turjanski

    Full Text Available 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 t(½, 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.

  2. Production of chicken chimeras by fusing blastodermal cells with electroporation

    Institute of Scientific and Technical Information of China (English)

    S.Aritomi; N.Fujihara

    2000-01-01

    Aim: To establish techniques for producing somatic and gennline chimeric chicken by transferring blastodennal cells fused with electroporation. Methods: Stage-X blastodermal cells isolated from freshly laid fertile unincubated white Leghom and Rhode Island red chicken eggs were fused with electroporation. The treated cell suspension was transferred to the recovery medium (DMEM containing 10% FBS) and was injected into the subgerminal cavity of recipient tmincubated embryos (stage X). Results: Of 177 recipient embryos injected with the fusing blastodermal cells, 6 (3.4%) survived to hatching. Somatic chimerism was examined in the melanocyte of the feather. The presence of feathers originating from the donor cell was observed in 1 bird (16.7%) out of the 6 hatched birds. After 21 days of incubation two birds out of five embryos were subjected to polymemse chain reaction (PCR) analysis for W-chromosome-specific DNA for each tissue. One bird possessed W-chromosome-specific DNA in the stomach, and the other exhibited the same DNA in the left and right gonads and other tissues, but not the stomach. Conclusion: Recipient embryo having electrofused blastodermal cells yields somatic and germline chimeric chickens more successfully.(Asian J Androl 2000 Dec;2:271-275)

  3. Endovascular nonthermal irreversible electroporation: a finite element analysis.

    Science.gov (United States)

    Maor, Elad; Rubinsky, Boris

    2010-03-01

    Tissue ablation finds an increasing use in modern medicine. Nonthermal irreversible electroporation (NTIRE) is a biophysical phenomenon and an emerging novel tissue ablation modality, in which electric fields are applied in a pulsed mode to produce nanoscale defects to the cell membrane phospholipid bilayer, in such a way that Joule heating is minimized and thermal damage to other molecules in the treated volume is reduced while the cells die. Here we present a two-dimensional transient finite element model to simulate the electric field and thermal damage to the arterial wall due to an endovascular NTIRE novel device. The electric field was used to calculate the Joule heating effect, and a transient solution of the temperature is presented using the Pennes bioheat equation. This is followed by a kinetic model of the thermal damage based on the Arrhenius formulation and calculation of the Henriques and Moritz thermal damage integral. The analysis shows that the endovascular application of 90, 100 mus pulses with a potential difference of 600 V can induce electric fields of 1000 V/cm and above across the entire arterial wall, which are sufficient for irreversible electroporation. The temperature in the arterial wall reached a maximum of 66.7 degrees C with a pulse frequency of 4 Hz. Thermal damage integral showed that this protocol will thermally damage less than 2% of the molecules around the electrodes. In conclusion, endovascular NTIRE is possible. Our study sets the theoretical basis for further preclinical and clinical trials with endovascular NTIRE.

  4. Transient transformation of Podosphaera xanthii by electroporation of conidia.

    Science.gov (United States)

    Vela-Corcía, David; Romero, Diego; Torés, Juan Antonio; De Vicente, Antonio; Pérez-García, Alejandro

    2015-02-06

    Powdery mildew diseases are a major phytosanitary issue causing important yield and economic losses in agronomic, horticultural and ornamental crops. Powdery mildew fungi are obligate biotrophic parasites unable to grow on culture media, a fact that has significantly limited their genetic manipulation. In this work, we report a protocol based on the electroporation of fungal conidia, for the transient transformation of Podosphaera fusca (synonym Podosphaera xanthii), the main causal agent of cucurbit powdery mildew. To introduce DNA into P. xanthii conidia, we applied two square-wave pulses of 1.7 kV for 1 ms with an interval of 5 s. We tested these conditions with several plasmids bearing as selective markers hygromycin B resistance (hph), carbendazim resistance (TUB2) or GFP (gfp) under control of endogenous regulatory elements from Aspergillus nidulans, Neurospora crassa or P. xanthii to drive their expression. An in planta selection procedure using the MBC fungicide carbendazim permitted the propagation of transformants onto zucchini cotyledons and avoided the phytotoxicity associated with hygromycin B. This is the first report on the transformation of P. xanthii and the transformation of powdery mildew fungi using electroporation. Although the transformants are transient, this represents a feasible method for the genetic manipulation of this important group of plant pathogens.

  5. Efficient transformation of Rhizopus delemar by electroporation of germinated spores.

    Science.gov (United States)

    Xu, Sha; Zhou, Zhengxiong; Du, Guocheng; Zhou, Jingwen; Chen, Jian

    2014-08-01

    High efficient transformation of mycelial fungi is essential to both metabolic engineering and physiological analysis of these industrially important microorganisms. However, transformation efficiencies for mycelial fungi are highly restricted by difficulties in colony formation and competent cell preparation. In this work, an innovative transformation procedure that could significantly improve the efficiency of colony formation and transformation process has been established for a typical mycelial fungus, Rhizopus delemar. Single colonies of R. delemar were obtained with the addition of sodium deoxycholate. Fresh germinated spores of R. delemar were successfully transformed by electroporation. In addition, by pretreatment of the germinated spores with 0.05M lithium acetate (LiAc) and 20mM dithiothreitol (DTT) before electroporation, the transformation efficiency was further improved by 9.5-fold. The final transformation efficiency at optimal conditions reached 1239 transformants/μg DNA. The method described here would facilitate more efficient metabolic engineering and investigation of physiological functions in R. delemar or other similar mycelial fungi.

  6. Cell electroporation with a three-dimensional microelectrode array on a printed circuit board.

    Science.gov (United States)

    Xu, Youchun; Su, Shisheng; Zhou, Changcheng; Lu, Ying; Xing, Wanli

    2015-04-01

    Electroporation is a commonly used approach to rapidly introduce exogenous molecules into cells without permanent damage. Compared to classical electroporation protocols, microchip-based electroporation approaches have the advantages of high transfection efficiency and low consumption, but they also commonly rely on costly and tedious microfabrication technology. Hence, it is desirable to develop a novel, more affordable, and effective approach to facilitate cell electroporation. In this study, we utilized a standard printed circuit board (PCB) technology to fabricate a chip with an interdigitated array of electrodes for electroporation of suspended cells. The electrodes (thickness ~35 μm) fabricated by PCB technology are much thicker than the two-dimensional (2D) planar electrodes (thickness electroporation. HeLa, MCF7, COS7, Jurkat, and 3T3-L1 cells were efficiently transfected with the pEGFP-N1 plasmid using individually optimal electroporation parameters. This work provides a novel method for convenient and rapid cell transfection and thus holds promise for use as a low-cost disposable device in biomedical research.

  7. Method for electric parametric characterization and optimization of electroporation on a chip.

    Science.gov (United States)

    Wu, Mengxi; Zhao, Deyao; Wei, Zewen; Zhong, Wenfeng; Yan, Hao; Wang, Xiaoxia; Liang, Zicai; Li, Zhihong

    2013-05-01

    We have developed a rapid method to optimize the electric parameters of cell electroporation. In our design, a pair of ring-dot formatted electrodes was used to generate a radial distribution of electric field from the center to the periphery. Varied electric field intensity was acquired in different annulus when an electric pulse was applied. Cells were cultured on the microchips for adherent cell electroporation and in situ observation. The electroporation parameters of electric field intensity were explored and evaluated in terms of cell viability and transfection efficiency. The optimization was performed in consideration of both cell viability, which was investigated to decrease as electric field increases, and the transfection rate, which normally increases at stronger electric field. The electroporation characteristics HEK-293A and Hela cells were investigated, and the optimum parameters were obtained. Verified by a commercial electroporation system as well as self-made microchips endowed the optimization with wider meaning. At last, as applications, we acquired the optimal electroporation pulse intensity of Neuro-2A cells and a type of primary cell (human umbilical vein endothelial cell, HUVEC) by one time electroporation using the proposed method.

  8. Electroporation transiently decreases GJB2 (connexin 26) expression in B16/BL6 melanoma cell line.

    Science.gov (United States)

    Rangel, Marcelo Monte Mór; Chaible, Lucas Martins; Nagamine, Marcia Kazumi; Mennecier, Gregory; Cogliati, Bruno; de Oliveira, Krishna Duro; Fukumasu, Heidge; Sinhorini, Idércio Luiz; Mir, Lluis Maria; Dagli, Maria Lúcia Zaidan

    2015-02-01

    Connexins are proteins that form gap junctions. Perturbations in the cell membrane reportedly promote changes in the expression profile of connexins. Electroporation promotes destabilization by applying electrical pulses, and this procedure is used in electrochemotherapy and gene therapy, among others. This in vitro work aimed to study the interference of electroporation on the expression profile of GJB2 (Cx26 gene) and Connexin 26 in melanoma cell line B16/BL6. The techniques of immunocytochemistry, Western blot, and real-time PCR were used. After electroporation, cells showed a transient decrease in GJB2 mRNA. The immunostaining of Cx26 showed no noticeable change after electroporation at different time points. However, Western blot showed a significant reduction in Cx26 30 min after electroporation. Our results showed that electroporation interferes transiently in the expression of Connexin 26 in melanoma and are consistent with the idea that electroporation is a process of intense stress that promotes cell homeostatic imbalance and results in disruption of cell physiological processes such as transcription and translation.

  9. Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in Xenopus

    Directory of Open Access Journals (Sweden)

    Piper Michael

    2007-09-01

    Full Text Available Abstract Background Blastomere injection of mRNA or antisense oligonucleotides has proven effective in analyzing early gene function in Xenopus. However, functional analysis of genes involved in neuronal differentiation and axon pathfinding by this method is often hampered by earlier function of these genes during development. Therefore, fine spatio-temporal control of over-expression or knock-down approaches is required to specifically address the role of a given gene in these processes. Results We describe here an electroporation procedure that can be used with high efficiency and low toxicity for targeting DNA and antisense morpholino oligonucleotides (MOs into spatially restricted regions of the Xenopus CNS at a critical time-window of development (22–50 hour post-fertilization when axonal tracts are first forming. The approach relies on the design of "electroporation chambers" that enable reproducible positioning of fixed-spaced electrodes coupled with accurate DNA/MO injection. Simple adjustments can be made to the electroporation chamber to suit the shape of different aged embryos and to alter the size and location of the targeted region. This procedure can be used to electroporate separate regions of the CNS in the same embryo allowing separate manipulation of growing axons and their intermediate and final targets in the brain. Conclusion Our study demonstrates that electroporation can be used as a versatile tool to investigate molecular pathways involved in axon extension during Xenopus embryogenesis. Electroporation enables gain or loss of function studies to be performed with easy monitoring of electroporated cells. Double-targeted transfection provides a unique opportunity to monitor axon-target interaction in vivo. Finally, electroporated embryos represent a valuable source of MO-loaded or DNA transfected cells for in vitro analysis. The technique has broad applications as it can be tailored easily to other developing organ

  10. Large-Scale mRNA Transfection of Dendritic Cells by Electroporation in Continuous Flow Systems

    DEFF Research Database (Denmark)

    Selmeczi, Dávid; Hansen, Thomas Steen; Met, Özcan

    2016-01-01

    Electroporation is well established for transient mRNA transfection of many mammalian cells, including immune cells such as dendritic cells used in cancer immunotherapy. Therapeutic application requires methods to efficiently electroporate and transfect millions of immune cells in a fast process...... the instrumentation and methods needed for the efficient transfection by electroporation of millions of dendritic cells in one continuous flow process....... with high cell survival. Continuous flow of suspended dendritic cells through a channel incorporating spatially separated microporous meshes with a synchronized electrical pulsing sequence can yield dendritic cell transfection rates of >75 % with survival rates of >90 %. This chapter describes...

  11. ELECTROPORATION OF CONFLUENT HM-1 ES CELLS LEADS TO HIGHER AMOUNT COLONIES

    Directory of Open Access Journals (Sweden)

    A.ALENA BENCSIK

    2013-12-01

    Full Text Available Electroporation is used to induce homologous recombination in the genome of the murine ES (embryonic stem cells. Routinelly subconfluent ES cells are recommended to be used in such experiments. Electroporation of immunoglobulin specific targeting vectors with different length of homology leads to reduced number of selected colonies. The enrichment of double selected colonies is high and thus the amount of HM-1 ES cell colonies for the analysis is very low. Here we show that the electroporation of confluent HM-1 ES cells leads to an increased amount of simple and double selected colonies.

  12. Localized electroporation effect on adherent cells in modified electric cell-substrate impedance sensing circuits

    Science.gov (United States)

    Kim, Yu Jin; Ram Song, Ka; Kim, Hee-Dae; Park, Bum Chul; Kim, Young Keun; Kang, Chi Jung

    2016-10-01

    Electroporation is a physical transfection method for introducing foreign genes or drugs into cells. It does not require toxic reagents or transfection vectors. However, its applications have been limited because of cell damage and nonspecific transport. Here, we present an effective method for selective and localized electroporation using atomic force microscopy. This electroporation method is applied to adherent cells on substrates, instead of conventionally used suspended cells, and offers relatively effective cell transfection. Moreover, this method enables localized transfection into targeted areas at the single-cell level.

  13. Optimising Code Generation with haggies

    OpenAIRE

    Reiter, Thomas

    2009-01-01

    This article describes haggies, a program for the generation of optimised programs for the efficient numerical evaluation of mathematical expressions. It uses a multivariate Horner-scheme and Common Subexpression Elimination to reduce the overall number of operations. The package can serve as a back-end for virtually any general purpose computer algebra program. Built-in type inference that allows to deal with non-standard data types in strongly typed languages and a very flexible, pattern-ba...

  14. Direct Gene Transfer into Plant Mature Seeds via Electroporation After Vacuum Treatment

    Science.gov (United States)

    Hagio, Takashi

    A number of direct gene transfer methods have been used successfully in plant genetic engineering, providing powerful tools to investigate fundamental and applied problems in plant biology (Chowrira et al., 1996; D'halluin et al., 1992; Morandini and Salamini, 2003; Rakoczy-Trojanowska, 2002; Songstad et al., 1995). In cereals, several methods have been found to be suitable for obtaining transgenic plant; these include bombardment of scutellum (Hagio et al., 1995) and inflorescence cultures (He et al., 2001), and silicon carbide fiber-mediated DNA delivery (Asano et al., 1991) and Agrobacterium tumefaciens transformation (Potrykus, 1990). Electroporation of cereal protoplasts also has proved successful but it involves prolonged cell treatments and generally is limited by the difficulties of regeneration from cereal protoplast cultures (Fromm et al., 1987). Many laboratories worldwide are now using Agrobacterium as a vehicle for routine production of transgenic crop plants. The primary application of the particle system (Klein et al., 1987) has been for transformation of species recalcitrant to conventional Agrobacterium (Binns, 1990) or protoplast methods. But these conventional methods can be applied to the species and varieties that are amenable to tissue culture (Machii et al., 1998). Mature seeds are readily available and free from the seasonal limits that immature embryo, inflorescence, and anther have. This method enables us to produce transgenic plants without time-consuming tissue culture process.

  15. In vivo electroporation enhances the immunogenicity of an HIV-1 DNA vaccine candidate in healthy volunteers.

    Directory of Open Access Journals (Sweden)

    Sandhya Vasan

    Full Text Available BACKGROUND: DNA-based vaccines have been safe but weakly immunogenic in humans to date. METHODS AND FINDINGS: We sought to determine the safety, tolerability, and immunogenicity of ADVAX, a multigenic HIV-1 DNA vaccine candidate, injected intramuscularly by in vivo electroporation (EP in a Phase-1, double-blind, randomized placebo-controlled trial in healthy volunteers. Eight volunteers each received 0.2 mg, 1 mg, or 4 mg ADVAX or saline placebo via EP, or 4 mg ADVAX via standard intramuscular injection at weeks 0 and 8. A third vaccination was administered to eleven volunteers at week 36. EP was safe, well-tolerated and considered acceptable for a prophylactic vaccine. EP delivery of ADVAX increased the magnitude of HIV-1-specific cell mediated immunity by up to 70-fold over IM injection, as measured by gamma interferon ELISpot. The number of antigens to which the response was detected improved with EP and increasing dosage. Intracellular cytokine staining analysis of ELISpot responders revealed both CD4+ and CD8+ T cell responses, with co-secretion of multiple cytokines. CONCLUSIONS: This is the first demonstration in healthy volunteers that EP is safe, tolerable, and effective in improving the magnitude, breadth and durability of cellular immune responses to a DNA vaccine candidate. TRIAL REGISTRATION: ClinicalTrials.gov NCT00545987.

  16. Efficient in vivo electroporation of the postnatal rodent forebrain.

    Directory of Open Access Journals (Sweden)

    Camille Boutin

    Full Text Available Functional gene analysis in vivo represents still a major challenge in biomedical research. Here we present a new method for the efficient introduction of nucleic acids into the postnatal mouse forebrain. We show that intraventricular injection of DNA followed by electroporation induces strong expression of transgenes in radial glia, neuronal precursors and neurons of the olfactory system. We present two proof-of-principle experiments to validate our approach. First, we show that expression of a human isoform of the neural cell adhesion molecule (hNCAM-140 in radial glia cells induces their differentiation into cells showing a neural precursor phenotype. Second, we demonstrate that p21 acts as a cell cycle inhibitor for postnatal neural stem cells. This approach will represent an important tool for future studies of postnatal neurogenesis and of neural development in general.

  17. 96-well electroporation method for transfection of mammalian central neurons.

    Science.gov (United States)

    Buchser, William J; Pardinas, Jose R; Shi, Yan; Bixby, John L; Lemmon, Vance P

    2006-11-01

    Manipulating gene expression in primary neurons has been a goal for many scientists for over 20 years. Vertebrate central nervous system neurons are classically difficult to transfect. Most lipid reagents are inefficient and toxic to the cells, and time-consuming methods such as viral infections are often required to obtain better efficiencies. We have developed an efficient method for the transfection of cerebellar granule neurons and hippocampal neurons with standard plasmid vectors. Using 96-well electroporation plates, square-wave pulses can introduce 96 different plasmids into neurons in a single step. The procedure results in greater than 20% transfection efficiencies and requires only simple solutions of nominal cost. In addition to enabling the rapid optimization of experimental protocols with multiple parameters, this procedure enables the use of high content screening methods to characterize neuronal phenotypes.

  18. Electroporation-enhanced delivery of nucleic acid vaccines.

    Science.gov (United States)

    Broderick, Kate E; Humeau, Laurent M

    2015-02-01

    The naked delivery of nucleic acid vaccines is notoriously inefficient, and an enabling delivery technology is required to direct efficiently these constructs intracellularly. A delivery technology capable of enhancing nucleic acid uptake in both cells in tissues and in culture is electroporation (EP). EP is a physical delivery mechanism that increases the permeability of mammalian cell membranes and allows the trafficking of large macromolecules into the cell. EP has now been used extensively in the clinic and been shown to be an effective method to increase both the uptake of the construct and the breadth and magnitude of the resulting immune responses. Excitingly, 2014 saw the announcement of the first EP-enhanced DNA vaccine Phase II trial demonstrating clinical efficacy. This review seeks to introduce the reader to EP as a technology to enhance the delivery of DNA and RNA vaccines and highlight several published clinical trials using this delivery modality.

  19. Electroporation of Chlorella vulgaris to enhance biomethane production.

    Science.gov (United States)

    Garoma, Temesgen; Shackelford, Trevor

    2014-10-01

    This research investigated the feasibility of using electroporation (EP) as a pretreatment method for algal biomass used as feedstock for anaerobic digestion. The results showed that pretreating algal biomass with EP significantly improved the soluble COD (SCOD), increasing it to more than 830% at 28 kWh/m(3) treatment intensity (TI). Besides TI, culture conditions also affected the performance of the EP process. On the basis of SCOD, a sample pH of 7.0 and cell concentration of 13.2g/L were found to be optimal for the EP process. Despite a direct relationship between TI and ionic strength (IS), SCOD decreased with increasing IS. At 35 kWh/m(3) TI, bio-CH4 production increased by as high as 110%. It was also observed that lower TI levels resulted in high rates of gain per energy input compared to higher degrees of treatment. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Focal Therapy of Prostate Cancer Using Irreversible Electroporation.

    Science.gov (United States)

    Valerio, Massimo; Ahmed, Hashim U; Emberton, Mark

    2015-09-01

    Focal therapy is a novel strategy that attempts to enhance the therapeutic ratio of standard radical treatment in prostate cancer. Irreversible electroporation (IRE) has some inherent characteristics that may be ideal for focal therapy. Precise confined ablation in the treatment area obtained via nonthermal damage with potential for minimal toxicity to surrounding structures may lead to optimal treatment with improved preservation of continence and erectile function. Initial data of focal IRE of the prostate are encouraging although further assessment is awaited to confirm these findings using robust methodology. In this article, we provide a comprehensive step-by-step description of our technique to deliver focal IRE in selected men with localized prostate cancer located in a discrete area of the prostate.

  1. Electroporation enhances mitomycin C cytotoxicity on T24 bladder cancer cell line

    DEFF Research Database (Denmark)

    Vasquez, Juan Luis; Gehl, Julie; Hermann, Gregers G

    2012-01-01

    Intravesical mitomycin instillation combined with electric pulses is being used experimentally for the treatment of T1 bladder tumors, in patients unfit for surgery. Electroporation may enhance the uptake of chemotherapeutics by permeabilization of cell membranes. We investigated if electroporati...

  2. What you always needed to know about electroporation based DNA vaccines

    DEFF Research Database (Denmark)

    Gothelf, Anita Birgitte; Gehl, Julie

    2012-01-01

    Vaccinations are increasingly used to fight infectious disease, and DNA vaccines offer considerable advantages, including broader possibilities for vaccination and lack of need for cold storage. It has been amply demonstrated, that electroporation augments uptake of DNA in both skin and muscle......, and it is foreseen that future DNA vaccination may to a large extent be coupled with and dependent upon electroporation based delivery. Understanding the basic science of electroporation and exploiting knowledge obtained on optimization of DNA electrotransfer to muscle and skin, may greatly augment efforts...... on vaccine development. The purpose of this review is to give a succinct but comprehensive overview of electroporation as a delivery modality including electrotransfer to skin and muscle. As well, this review will speculate and discuss future uses for this powerful electrotransfer technology....

  3. Intratumoral DNA electroporation induces anti-tumor immunity and tumor regression.

    Science.gov (United States)

    Radkevich-Brown, Olga; Piechocki, Marie P; Back, Jessica B; Weise, Amy M; Pilon-Thomas, Shari; Wei, Wei-Zen

    2010-03-01

    In situ expression of a foreign antigen and an immune-modulating cytokine by intratumoral DNA electroporation was tested as a cancer therapy regimen. Transgene expression in the tumors was sustained for 2-3 weeks after intratumoral electroporation with mammalian expression plasmid containing firefly luciferase cDNA. Electroporation with cDNA encoding tetanus toxin fragment C (TetC) induced tetanus toxin-binding antibody, demonstrating immune recognition of the transgene product. Intratumoral electroporation with TetC and IL-12 cDNA after mice were treated with CD25 mAb to remove regulatory T cells induced IFN-gamma producing T-cell response to tumor-associated antigen, heavy inflammatory infiltration, regression of established tumors and immune memory to protect mice from repeated tumor challenge. Intratumoral expression of immune-modulating molecules may be most suitable in the neoadjuvant setting to enhance the therapeutic efficacy and provide long-term protection.

  4. Lightning-triggered electroporation and electrofusion as possible contributors to natural HGT among prokaryotes

    CERN Document Server

    Kotnik, Tadej

    2012-01-01

    Phylogenetic studies show that horizontal gene transfer (HGT) is a significant contributor to genetic variability of prokaryotes, and was perhaps even more abundant during early evolution. Hitherto, research of natural HGT has mainly focused on three mechanisms: conjugation, natural competence, and viral transduction. This paper discusses the feasibility of a fourth such mechanism - cell electroporation and/or electrofusion triggered by atmospheric electrostatic discharges (lightnings). A description of electroporation as a phenomenon is followed by a review of experimental evidence that electroporation of prokaryotes in aqueous environments can result in release of non-denatured DNA, as well as uptake of DNA from the surroundings and transformation. Similarly, a description of electrofusion is followed by a review of experiments showing that prokaryotes devoid of cell wall can electrofuse into hybrids expressing the genes of their both precursors. Under sufficiently fine-tuned conditions, electroporation and...

  5. Theoretical analysis of AC electric field transmission into biological tissue through frozen saline for electroporation.

    Science.gov (United States)

    Xiao, Chunyan; Rubinsky, Boris

    2014-12-01

    An analytical model was used to explore the feasibility of sinusoidal electric field transmission across a frozen saline layer into biological tissue. The study is relevant to electroporation and permeabilization of the cell membrane by electric fields. The concept was analyzed for frequencies in the range of conventional electroporation frequencies and electric field intensity. Theoretical analysis for a variety of tissues show that the transmission of electroporation type electric fields through a layer of frozen saline into tissue is feasible and the behavior of this composite system depends on tissue type, frozen domain temperature, and frequency. Freezing could become a valuable method for adherence of electroporation electrodes to moving tissue surfaces, such as the heart in the treatment of atrial fibrillation or blood vessels for the treatment of restenosis.

  6. Electroporation Enhanced Effect of Dystrophin Splice Switching PNA Oligomers in Normal and Dystrophic Muscle

    DEFF Research Database (Denmark)

    Hjortkjær, Camilla Brolin; Shiraishi, Takehiko; Hojman, Pernille;

    2015-01-01

    and dystrophic mdx mice with or without electroporation. At low, single PNA doses (1.5, 3, or 10 µg/TA), electroporation augmented the antisense exon skipping induced by an unmodified PNA by twofold to fourfold in healthy mouse muscle with optimized electric parameters, measured after 7 days. The PNA splice...... switching was detected at the RNA level up to 4 weeks after a single-dose treatment. In dystrophic muscles of the MDX mouse, electroporation increased the number of dystrophin-positive fibers about 2.5-fold at 2 weeks after a single PNA administration compared to injection only. In conclusion, we find...... that electroporation can enhance PNA antisense effects in muscle tissue....

  7. High-efficiency nuclear transformation of the diatom Phaeodactylum tricornutum by electroporation.

    Science.gov (United States)

    Zhang, Chunye; Hu, Hanhua

    2014-08-01

    We established a high-efficiency nuclear transformation method for the diatom Phaeodactylum tricornutum using an electroporation system. Based on a universal electroporation protocol, the conditions for the introduction of exogenous DNA including electric field strength and plasmid form were optimized. Following optimization, the diatom cells could be transformed with exogenous gene easily, the maximum transformation frequency obtained was 2.8×10(-5) cells. The cotransformation of P. tricornutum with a non-selective GUS gene together with the selectable resistance gene has also been achieved using our new method and found to be very efficient (up to 60%). The electroporation procedure described in this article offers a number of advantages, including simplicity, general utility, low-cost and high efficiency. The described method also provides some clue for developing electroporation transformation system in other eukaryotic microalgae.

  8. Optically transparent polymer devices for in situ assessment of cell electroporation.

    Science.gov (United States)

    Majhi, Amit Kumar; Thrivikraman, Greeshma; Basu, Bikramjit; Venkataraman, V

    2015-02-01

    In order to study cell electroporation in situ, polymer devices have been fabricated from poly-dimethyl siloxane with transparent indium tin oxide parallel plate electrodes in horizontal geometry. This geometry with cells located on a single focal plane at the interface of the bottom electrode allows a longer observation time in both transmitted bright-field and reflected fluorescence microscopy modes. Using propidium iodide (PI) as a marker dye, the number of electroporated cells in a typical culture volume of 10-100 μl was quantified in situ as a function of applied voltage from 10 to 90 V in a series of ~2-ms pulses across 0.5-mm electrode spacing. The electric field at the interface and device current was calculated using a model that takes into account bulk screening of the transient pulse. The voltage dependence of the number of electroporated cells could be explained using a stochastic model for the electroporation kinetics, and the free energy for pore formation was found to be 45.6 ± 0.5 kT at room temperature. With this device, the optimum electroporation conditions can be quickly determined by monitoring the uptake of PI marker dye in situ under the application of millisecond voltage pulses. The electroporation efficiency was also quantified using an ex situ fluorescence-assisted cell sorter, and the morphology of cultured cells was evaluated after the pulsing experiment. Importantly, the efficacy of the developed device was tested independently using two cell lines (C2C12 mouse myoblast cells and yeast cells) as well as in three different electroporation buffers (phosphate buffer saline, electroporation buffer and 10% glycerol).

  9. Method for Preparation and Electroporation of S. aureus and S. epidermidis.

    Science.gov (United States)

    Grosser, Melinda R; Richardson, Anthony R

    2016-01-01

    For bacterial species that are not known to be naturally competent, such as Staphylococcus aureus and Staphylococcus epidermidis, electroporation is an efficient method for introducing genetic material into the cell. The technique utilizes electrical pulses to transiently permeabilize bacterial cell membranes, which allows for the passage of plasmid DNA across the membranes. Here, we describe methods for preparing electrocompetent S. aureus and S. epidermidis cells and outline a procedure for electroporation of the prepared competent cells.

  10. Robust optimisation of railway crossing geometry

    Science.gov (United States)

    Wan, Chang; Markine, Valeri; Dollevoet, Rolf

    2016-05-01

    This paper presents a methodology for improving the crossing (frog) geometry through the robust optimisation approach, wherein the variability of the design parameters within a prescribed tolerance is included in the optimisation problem. Here, the crossing geometry is defined by parameterising the B-spline represented cross-sectional shape and the longitudinal height profile of the nose rail. The dynamic performance of the crossing is evaluated considering the variation of wheel profiles and track alignment. A multipoint approximation method (MAM) is applied in solving the optimisation problem of minimising the contact pressure during the wheel-rail contact and constraining the location of wheel transition at the crossing. To clarify the difference between the robust optimisation and the normal deterministic optimisation approaches, the optimisation problems are solved in both approaches. The results show that the deterministic optimum fails under slight change of the design variables; the robust optimum, however, has improved and robust performance.

  11. Effects of deformability and thermal motion of lipid membrane on electroporation: By molecular dynamics simulations

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Sheng; Yin, Guangyao [Bioengineering Graduate Program, Hong Kong University of Science and Technology, Hong Kong Special Administrative Region (China); Lee, Yi-Kuen [Department of Mechanical Engineering, Hong Kong University of Science and Technology, Hong Kong Special Administrative Region (China); Wong, Joseph T.Y. [Division of Life Science, Hong Kong University of Science and Technology, Hong Kong Special Administrative Region (China); Zhang, Tong-Yi, E-mail: mezhangt@ust.hk [Department of Mechanical Engineering, Hong Kong University of Science and Technology, Hong Kong Special Administrative Region (China)

    2011-01-14

    Research highlights: {yields} MD simulations show that deformability and thermal motion of membrane affect electroporation. {yields} Stiffer membrane inhibits electroporation and makes water penetrate from both sides. {yields} Higher temperature accelerates electroporation. -- Abstract: Effects of mechanical properties and thermal motion of POPE lipid membrane on electroporation were studied by molecular dynamics simulations. Among simulations in which specific atoms of lipids were artificially constrained at their equilibrium positions using a spring with force constant of 2.0 kcal/(mol A{sup 2}) in the external electric field of 1.4 kcal/(mol A e), only constraint on lateral motions of lipid tails prohibited electroporation while non-tail parts had little effects. When force constant decreased to 0.2 kcal/(mol A{sup 2}) in the position constraints on lipid tails in the external electric field of 2.0 kcal/(mol A e), water molecules began to enter the membrane. Position constraints of lipid tails allow water to penetrate from both sides of membrane. Thermal motion of lipids can induce initial defects in the hydrophobic core of membrane, which are favorable nucleation sites for electroporation. Simulations at different temperatures revealed that as the temperature increases, the time taken to the initial pore formation will decrease.

  12. A novel electroporation system for efficient molecular delivery into Chlamydomonas reinhardtii with a 3-dimensional microelectrode

    Science.gov (United States)

    Kang, Seongsu; Kim, Kwon-Ho; Kim, Yeu-Chun

    2015-11-01

    Electroporation is one of the most widely used transfection methods because of its high efficiency and convenience among the various transfection methods. Previous micro-electroporation systems have some drawbacks such as limitations in height and design, time-consuming and an expensive fabrication process due to technical constraints. This study fabricates a three dimensional microelectrode using the 3D printing technique. The interdigitated microstructure consisting of poly lactic acid was injected by a 3D printer and coated with silver and aluminum with a series of dip-coatings. With the same strength of electric field (V cm-1), a higher efficiency for molecular delivery and a higher cellular viability are achieved with the microelectrode than with a standard cuvette. In addition, this study investigates chemicophysical changes such as Joule heating and dissolved metal during electroporation and showed the micro-electroporation system had less chemicophysical changes. It was concluded that the proposed micro-electroporation system will contribute to genetic engineering as a promising delivery tool, and this combination of 3D printing and electroporation has many potential applications for diverse designs or systems.

  13. Electroporation of cells using EM induction of ac fields by a magnetic stimulator

    Energy Technology Data Exchange (ETDEWEB)

    Chen, C; Robinson, M P [Department of Electronics, University of York, Heslington, York YO10 5DD (United Kingdom); Evans, J A [Academic Unit of Medical Physics, University of Leeds, Leeds LS2 9JT (United Kingdom); Smye, S W [Department of Medical Physics and Engineering, Leeds Teaching Hospitals, St. James' s University Hospital, Leeds LS9 7TF (United Kingdom); O' Toole, P [Department of Biology, University of York, Heslington, York YO10 5DD (United Kingdom)

    2010-02-21

    This paper describes a method of effectively electroporating mammalian cell membranes with pulsed alternating-current (ac) electric fields at field strengths of 30-160 kV m{sup -1}. Although many in vivo electroporation protocols entail applying square wave or monotonically decreasing pulses via needles or electrode plates, relatively few have explored the use of pulsed ac fields. Following our previous study, which established the effectiveness of ac fields for electroporating cell membranes, a primary/secondary coil system was constructed to produce sufficiently strong electric fields by electromagnetic induction. The primary coil was formed from the applicator of an established transcranial magnetic stimulation (TMS) system, while the secondary coil was a purpose-built device of a design which could eventually be implanted into tissue. The effects of field strength, pulse interval and cumulative exposure time were investigated using microscopy and flow cytometry. Results from experiments on concentrated cell suspensions showed an optimized electroporation efficiency of around 50%, demonstrating that electroporation can be practicably achieved by inducing such pulsed ac fields. This finding confirms the possibility of a wide range of in vivo applications based on magnetically coupled ac electroporation.

  14. Electroporation of proviral RCAS DNA alters gene expression in the embryonic chick hindbrain.

    Science.gov (United States)

    Hermann, Petra M; Logan, C Cairine

    2003-11-01

    Gene transfer by means of electroporation is an effective method for delivering DNA into cells. Expression vectors encoding green fluorescent protein (GFP) are routinely used as a control for this technique and are also regularly used to indirectly or directly monitor the expression of introduced transgenes. However, recent studies suggest that GFP may have nonspecific and/or cytotoxic side effects. In this study, we investigated the effects of enhanced GFP (EGFP) expression delivered by means of electroporation of proviral RCASBP(B)-EGFP DNA on gene expression in the hindbrain of chick embryos. We examined, via whole-mount in situ hybridization, the expression of a number of transcription factors. We found that Tlx-1 was ectopically expressed following electroporation of proviral RCASBP(B)-EGFP DNA. In contrast, the number of cells expressing Tlx-3, Phox2a, and Phox2b were reduced. Intriguingly, these effects could be mimicked by electroporation of wild-type proviral RCASBP(B) DNA (i.e., lacking the GFP insert). However, neither delivery of the EGFP transgene by means of viral infection nor electroporation alone yielded aberrant expression patterns. Together our data indicate that alterations of gene expression patterns are not directly due to the expression of EGFP but instead reflect a confounding effect of electroporating proviral DNA.

  15. A novel electroporation system for efficient molecular delivery into Chlamydomonas reinhardtii with a 3-dimensional microelectrode.

    Science.gov (United States)

    Kang, Seongsu; Kim, Kwon-Ho; Kim, Yeu-Chun

    2015-11-02

    Electroporation is one of the most widely used transfection methods because of its high efficiency and convenience among the various transfection methods. Previous micro-electroporation systems have some drawbacks such as limitations in height and design, time-consuming and an expensive fabrication process due to technical constraints. This study fabricates a three dimensional microelectrode using the 3D printing technique. The interdigitated microstructure consisting of poly lactic acid was injected by a 3D printer and coated with silver and aluminum with a series of dip-coatings. With the same strength of electric field (V cm(-1)), a higher efficiency for molecular delivery and a higher cellular viability are achieved with the microelectrode than with a standard cuvette. In addition, this study investigates chemicophysical changes such as Joule heating and dissolved metal during electroporation and showed the micro-electroporation system had less chemicophysical changes. It was concluded that the proposed micro-electroporation system will contribute to genetic engineering as a promising delivery tool, and this combination of 3D printing and electroporation has many potential applications for diverse designs or systems.

  16. Electroporation markedly improves Sleeping Beauty transposon-induced tumorigenesis in mice.

    Science.gov (United States)

    Jung, S; Choi, H-J; Park, H-K; Jo, W; Jang, S; Ryu, J-E; Kim, W-J; Yu, E-S; Son, W-C

    2014-08-01

    The Sleeping Beauty (SB) transposon system is an important tool for genetic studies. It is used to insert a gene of interest into the host chromosome, thus enabling permanent gene expression. However, this system is less useful in higher eukaryotes because the transposition frequency is low. Efforts to improve the efficacy of the SB transposon system have focused on the method of gene delivery, but although electroporation has recently attracted much attention as an in vivo gene delivery tool, the simultaneous use of electroporation and the SB transposon system has not been studied for gene transfer in mice. In this study, electroporation was used in a model of SB transposon-induced insertional tumorigenesis. Electroporation increased the rate of tumor development to three times that of the control group. There was no difference in phenotype between tumors induced with the SB transposon system alone and those induced by the SB transposon and electroporation. Electroporation therefore may be an efficient means of improving the efficacy of gene transfer via the SB transposon system.

  17. Generation of stable mutants and targeted gene deletion strains in Cryptococcus neoformans through electroporation.

    Science.gov (United States)

    Lin, Xiaorong; Chacko, Nadia; Wang, Linqi; Pavuluri, Yashwant

    2015-04-01

    Cryptococcus neoformans is the etiologic agent of cryptococcal meningitis that causes more than half a million deaths worldwide each year. This capsulated basidiomycetous yeast also serves as a model for micropathogenic studies. The ability to make stable mutants, either via ectopic integration or homologous recombination, has been accomplished using biolistic transformation. This technical advance has greatly facilitated the research on the basic biology and pathogenic mechanisms of this pathogen in the past two decades. However, biolistic transformation is costly, and its reproducibility varies widely. Here we found that stable ectopic integration or targeted gene deletion via homologous replacement could be accomplished through electroporative transformation. The stability of the transformants obtained through electroporation and the frequency of homologous replacement is highly dependent on the selective marker. A frequency of homologous recombination among the stable transformants obtained by electroporation is comparable to those obtained by biolistic transformation (∼10%) when dominant drug selection markers are used, which is much higher than what has been previously reported for electroporation when auxotrophic markers were used (0.001% to 0.1%). Furthermore, disruption of the KU80 gene or generation of gene deletion constructs using the split marker strategy, two approaches known to increase homologous replacement among transformants obtained through biolistic transformation, also increase the frequency of homologous replacement among transformants obtained through electroporation. Therefore, electroporation provides a low cost alternative for mutagenesis in Cryptococcus.

  18. An Optimisation Approach for Room Acoustics Design

    DEFF Research Database (Denmark)

    Holm-Jørgensen, Kristian; Kirkegaard, Poul Henning; Andersen, Lars

    2005-01-01

    This paper discuss on a conceptual level the value of optimisation techniques in architectural acoustics room design from a practical point of view. It is chosen to optimise one objective room acoustics design criterium estimated from the sound field inside the room. The sound field is modeled...... using the boundary element method where absorption is incorporated. An example is given where the geometry of a room is defined by four design modes. The room geometry is optimised to get a uniform sound pressure....

  19. Optimisation combinatoire Theorie et algorithmes

    CERN Document Server

    Korte, Bernhard; Fonlupt, Jean

    2010-01-01

    Ce livre est la traduction fran aise de la quatri me et derni re dition de Combinatorial Optimization: Theory and Algorithms crit par deux minents sp cialistes du domaine: Bernhard Korte et Jens Vygen de l'universit de Bonn en Allemagne. Il met l accent sur les aspects th oriques de l'optimisation combinatoire ainsi que sur les algorithmes efficaces et exacts de r solution de probl mes. Il se distingue en cela des approches heuristiques plus simples et souvent d crites par ailleurs. L ouvrage contient de nombreuses d monstrations, concises et l gantes, de r sultats difficiles. Destin aux tudia

  20. Optimisation of the LHCb detector

    CERN Document Server

    Hierck, R

    2003-01-01

    This thesis describes a comparison of the LHCb classic and LHCb light concept from a tracking perspective. The comparison includes the detector occupancies, the various pattern recognition algorithms and the reconstruction performance. The final optimised LHCb setup is used to study the physics performance of LHCb for the Bs->DsK and Bs->DsPi decay channels. This includes both the event selection and a study of the sensitivity for the Bs oscillation frequency, delta m_s, the Bs lifetime difference, DGamma_s, and the CP parameter gamma-2delta gamma.

  1. Optimisation of the LHCb detector

    CERN Document Server

    Hierck, R H

    2003-01-01

    This thesis describes a comparison of the LHCb classic and LHCb light concept from a tracking perspective. The comparison includes the detector occupancies, the various pattern recognition algorithms and the reconstruction performance. The final optimised LHCb setup is used to study the physics performance of LHCb for the Bs->DsK and Bs->DsPi decay channels. This includes both the event selection and a study of the sensitivity for the Bs oscillation frequency, delta m_s, the Bs lifetime difference, DGamma_s, and the CP parameter gamma-2delta gamma.

  2. Efficient large volume electroporation of dendritic cells through micrometer scale manipulation of flow in a disposable polymer chip

    DEFF Research Database (Denmark)

    Selmeczi, Dávid; Hansen, Thomas Steen; Met, Özcan

    2011-01-01

    of the micrometer sized holes in the meshes compared to the main channel enforces an almost homogeneous flow velocity between the meshes. Thereby, very uniform electroporation of the cells can be accomplished. Successful electroporation of 20 million human dendritic cells with mRNA is demonstrated. The performance...... of the chip is similar to that of the traditional electroporation cuvette, but without an upper limit on the number of cells to be electroporated. The device is constructed with two female Luer parts and can easily be integrated with other microfluidic components. Furthermore it is fabricated from injection...

  3. Optimising resource management in neurorehabilitation.

    Science.gov (United States)

    Wood, Richard M; Griffiths, Jeff D; Williams, Janet E; Brouwers, Jakko

    2014-01-01

    To date, little research has been published regarding the effective and efficient management of resources (beds and staff) in neurorehabilitation, despite being an expensive service in limited supply. To demonstrate how mathematical modelling can be used to optimise service delivery, by way of a case study at a major 21 bed neurorehabilitation unit in the UK. An automated computer program for assigning weekly treatment sessions is developed. Queue modelling is used to construct a mathematical model of the hospital in terms of referral submissions to a waiting list, admission and treatment, and ultimately discharge. This is used to analyse the impact of hypothetical strategic decisions on a variety of performance measures and costs. The project culminates in a hybridised model of these two approaches, since a relationship is found between the number of therapy hours received each week (scheduling output) and length of stay (queuing model input). The introduction of the treatment scheduling program has substantially improved timetable quality (meaning a better and fairer service to patients) and has reduced employee time expended in its creation by approximately six hours each week (freeing up time for clinical work). The queuing model has been used to assess the effect of potential strategies, such as increasing the number of beds or employing more therapists. The use of mathematical modelling has not only optimised resources in the short term, but has allowed the optimality of longer term strategic decisions to be assessed.

  4. Effects of in ovo electroporation on endogenous gene expression: genome-wide analysis

    Directory of Open Access Journals (Sweden)

    Chambers David

    2011-04-01

    Full Text Available Abstract Background In ovo electroporation is a widely used technique to study gene function in developmental biology. Despite the widespread acceptance of this technique, no genome-wide analysis of the effects of in ovo electroporation, principally the current applied across the tissue and exogenous vector DNA introduced, on endogenous gene expression has been undertaken. Here, the effects of electric current and expression of a GFP-containing construct, via electroporation into the midbrain of Hamburger-Hamilton stage 10 chicken embryos, are analysed by microarray. Results Both current alone and in combination with exogenous DNA expression have a small but reproducible effect on endogenous gene expression, changing the expression of the genes represented on the array by less than 0.1% (current and less than 0.5% (current + DNA, respectively. The subset of genes regulated by electric current and exogenous DNA span a disparate set of cellular functions. However, no genes involved in the regional identity were affected. In sharp contrast to this, electroporation of a known transcription factor, Dmrt5, caused a much greater change in gene expression. Conclusions These findings represent the first systematic genome-wide analysis of the effects of in ovo electroporation on gene expression during embryonic development. The analysis reveals that this process has minimal impact on the genetic basis of cell fate specification. Thus, the study demonstrates the validity of the in ovo electroporation technique to study gene function and expression during development. Furthermore, the data presented here can be used as a resource to refine the set of transcriptional responders in future in ovo electroporation studies of specific gene function.

  5. Electroporation facilitates introduction of reporter transgenes and virions into schistosome eggs.

    Directory of Open Access Journals (Sweden)

    Kristine J Kines

    2010-02-01

    Full Text Available The schistosome egg represents an attractive developmental stage at which to target transgenes because of the high ratio of germ to somatic cells, because the transgene might be propagated and amplified by infecting snails with the miracidia hatched from treated eggs, and because eggs can be readily obtained from experimentally infected rodents.We investigated the utility of square wave electroporation to deliver transgenes and other macromolecules including fluorescent (Cy3 short interference (si RNA molecules, messenger RNAs, and virions into eggs of Schistosoma mansoni. First, eggs were incubated in Cy3-labeled siRNA with and without square wave electroporation. Cy3-signals were detected by fluorescence microscopy in eggs and miracidia hatched from treated eggs. Second, electroporation was employed to introduce mRNA encoding firefly luciferase into eggs. Luciferase activity was detected three hours later, whereas luciferase was not evident in eggs soaked in the mRNA. Third, schistosome eggs were exposed to Moloney murine leukemia virus virions (MLV pseudotyped with vesicular stomatitis virus glycoprotein (VSVG. Proviral transgenes were detected by PCR in genomic DNA from miracidia hatched from virion-exposed eggs, indicating the presence of transgenes in larval schistosomes that had been either soaked or electroporated. However, quantitative PCR (qPCR analysis determined that electroporation of virions resulted in 2-3 times as many copies of provirus in these schistosomes compared to soaking alone. In addition, relative qPCR indicated a copy number for the proviral luciferase transgene of approximately 20 copies for 100 copies of a representative single copy endogenous gene (encoding cathepsin D.Square wave electroporation facilitates introduction of transgenes into the schistosome egg. Electroporation was more effective for the transduction of eggs with pseudotyped MLV than simply soaking the eggs in virions. These findings underscore the

  6. Efficient transfection of DNA into primarily cultured rat sertoli cells by electroporation.

    Science.gov (United States)

    Li, Fuping; Yamaguchi, Kohei; Okada, Keisuke; Matsushita, Kei; Enatsu, Noritoshi; Chiba, Koji; Yue, Huanxun; Fujisawa, Masato

    2013-03-01

    The expression of exogenous DNA in Sertoli cells is essential for studying its functional genomics, pathway analysis, and medical applications. Electroporation is a valuable tool for nucleic acid delivery, even in primarily cultured cells, which are considered difficult to transfect. In this study, we developed an optimized protocol for electroporation-based transfection of Sertoli cells and compared its efficiency with conventional lipofection. Sertoli cells were transfected with pCMV-GFP plasmid by square-wave electroporation under different conditions. After transfection of plasmid into Sertoli cells, enhanced green fluorescent protein (EGFP) expression could be easily detected by fluorescent microscopy, and cell survival was evaluated by dye exclusion assay using Trypan blue. In terms of both cell survival and the percentage expressing EGFP, 250 V was determined to produce the greatest number of transiently transfected cells. Keeping the voltage constant (250 V), relatively high cell survival (76.5% ± 3.4%) and transfection efficiency (30.6% ± 5.6%) were observed with a pulse length of 20 μm. The number of pulses significantly affected cell survival and EGFP expression (P electroporation (21.5% ± 5.7%) was significantly higher than those of Lipofectamine 2000 (2.9% ± 1.0%) and Effectene (1.9% ± 0.8%) in this experiment (P electroporation conditions, and the successful electroporation of plasmid DNA into primarily cultured Sertoli cells. Our results indicate that the method of electroporation is more suitable than other approaches for the transfection of Sertoli cells.

  7. Dual-porosity model of solute diffusion in biological tissue modified by electroporation.

    Science.gov (United States)

    Mahnič-Kalamiza, Samo; Miklavčič, Damijan; Vorobiev, Eugène

    2014-07-01

    In many electroporation applications mass transport in biological tissue is of primary concern. This paper presents a theoretical advancement in the field and gives some examples of model use in electroporation applications. The study focuses on post-treatment solute diffusion. We use a dual-porosity approach to describe solute diffusion in electroporated biological tissue. The cellular membrane presents a hindrance to solute transport into the extracellular space and is modeled as electroporation-dependent porosity, assigned to the intracellular space (the finite rate of mass transfer within an individual cell is not accounted for, for reasons that we elaborate on). The second porosity is that of the extracellular space, through which solute vacates a block of tissue. The model can be used to study extraction out of or introduction of solutes into tissue, and we give three examples of application, a full account of model construction, validation with experiments, and a parametrical analysis. To facilitate easy implementation and experimentation by the reader, the complete derivation of the analytical solution for a simplified example is presented. Validation is done by comparing model results to experimentally-obtained data; we modeled kinetics of sucrose extraction by diffusion from sugar beet tissue in laboratory-scale experiments. The parametrical analysis demonstrates the importance of selected physicochemical and geometrical properties of the system, illustrating possible outcomes of applying the model to different electroporation applications. The proposed model is a new platform that supports rapid extension by state-of-the-art models of electroporation phenomena, developed as latest achievements in the field of electroporation.

  8. Dose optimisation in single plane interstitial brachytherapy

    DEFF Research Database (Denmark)

    Tanderup, Kari; Hellebust, Taran Paulsen; Honoré, Henriette Benedicte;

    2006-01-01

    BACKGROUND AND PURPOSE: Brachytherapy dose distributions can be optimised       by modulation of source dwell times. In this study dose optimisation in       single planar interstitial implants was evaluated in order to quantify the       potential benefit in patients. MATERIAL AND METHODS: In 14...

  9. Self-optimising control of sewer systems

    DEFF Research Database (Denmark)

    Mauricio Iglesias, Miguel; Montero-Castro, Ignacio; Mollerup, Ane Loft;

    The design of sewer system control is a complex task given the large size of the sewer networks, the transient dynamics of the water flows and the stochastic nature of rainfall. This contribution presents a generic methodology for the design of a self-optimising controller in sewer systems...... to design an optimising control strategy for a subcathcment area in Copenhagen....

  10. An Optimisation Approach for Room Acoustics Design

    DEFF Research Database (Denmark)

    Holm-Jørgensen, Kristian; Kirkegaard, Poul Henning; Andersen, Lars

    2005-01-01

    This paper discuss on a conceptual level the value of optimisation techniques in architectural acoustics room design from a practical point of view. It is chosen to optimise one objective room acoustics design criterium estimated from the sound field inside the room. The sound field is modeled...

  11. Haemodynamic optimisation in lower limb arterial surgery

    DEFF Research Database (Denmark)

    Bisgaard, J; Gilsaa, T; Rønholm, E;

    2012-01-01

    index was optimised by administering 250 ml aliquots of colloid intraoperatively and during the first 6 h post-operatively. Following surgery, fluid optimisation was supplemented with dobutamine, if necessary, targeting an oxygen delivery index level ≥ 600 ml/min(/) m(2) in the intervention group...

  12. Evolutionary programming for neutron instrument optimisation

    Science.gov (United States)

    Bentley, Phillip M.; Pappas, Catherine; Habicht, Klaus; Lelièvre-Berna, Eddy

    2006-11-01

    Virtual instruments based on Monte-Carlo techniques are now integral part of novel instrumentation development and the existing codes (McSTAS and Vitess) are extensively used to define and optimise novel instrumental concepts. Neutron spectrometers, however, involve a large number of parameters and their optimisation is often a complex and tedious procedure. Artificial intelligence algorithms are proving increasingly useful in such situations. Here, we present an automatic, reliable and scalable numerical optimisation concept based on the canonical genetic algorithm (GA). The algorithm was used to optimise the 3D magnetic field profile of the NSE spectrometer SPAN, at the HMI. We discuss the potential of the GA which combined with the existing Monte-Carlo codes (Vitess, McSTAS, etc.) leads to a very powerful tool for automated global optimisation of a general neutron scattering instrument, avoiding local optimum configurations.

  13. Evolutionary programming for neutron instrument optimisation

    Energy Technology Data Exchange (ETDEWEB)

    Bentley, Phillip M. [Hahn-Meitner Institut, Glienicker Strasse 100, D-14109 Berlin (Germany)]. E-mail: phillip.bentley@hmi.de; Pappas, Catherine [Hahn-Meitner Institut, Glienicker Strasse 100, D-14109 Berlin (Germany); Habicht, Klaus [Hahn-Meitner Institut, Glienicker Strasse 100, D-14109 Berlin (Germany); Lelievre-Berna, Eddy [Institut Laue-Langevin, 6 rue Jules Horowitz, BP 156, 38042 Grenoble Cedex 9 (France)

    2006-11-15

    Virtual instruments based on Monte-Carlo techniques are now integral part of novel instrumentation development and the existing codes (McSTAS and Vitess) are extensively used to define and optimise novel instrumental concepts. Neutron spectrometers, however, involve a large number of parameters and their optimisation is often a complex and tedious procedure. Artificial intelligence algorithms are proving increasingly useful in such situations. Here, we present an automatic, reliable and scalable numerical optimisation concept based on the canonical genetic algorithm (GA). The algorithm was used to optimise the 3D magnetic field profile of the NSE spectrometer SPAN, at the HMI. We discuss the potential of the GA which combined with the existing Monte-Carlo codes (Vitess, McSTAS, etc.) leads to a very powerful tool for automated global optimisation of a general neutron scattering instrument, avoiding local optimum configurations.

  14. The effects of irreversible electroporation (IRE on nerves.

    Directory of Open Access Journals (Sweden)

    Wei Li

    Full Text Available BACKGROUND: If a critical nerve is circumferentially involved with tumor, radical surgery intended to cure the cancer must sacrifice the nerve. Loss of critical nerves may lead to serious consequences. In spite of the impressive technical advancements in nerve reconstruction, complete recovery and normalization of nerve function is difficult to achieve. Though irreversible electroporation (IRE might be a promising choice to treat tumors near or involved critical nerve, the pathophysiology of the nerve after IRE treatment has not be clearly defined. METHODS: We applied IRE directly to a rat sciatic nerve to study the long term effects of IRE on the nerve. A sequence of 10 square pulses of 3800 V/cm, each 100 µs long was applied directly to rat sciatic nerves. In each animal of group I (IRE the procedure was applied to produce a treated length of about 10 mm. In each animal of group II (Control the electrodes were only applied directly on the sciatic nerve for the same time. Electrophysiological, histological, and functional studies were performed on immediately after and 3 days, 1 week, 3, 5, 7 and 10 weeks following surgery. FINDINGS: Electrophysiological, histological, and functional results show the nerve treated with IRE can attain full recovery after 7 weeks. CONCLUSION: This finding is indicative of the preservation of nerve involving malignant tumors with respect to the application of IRE pulses to ablate tumors completely. In summary, IRE may be a promising treatment tool for any tumor involving nerves.

  15. Transformation of Epichloë typhina by electroporation of conidia

    Directory of Open Access Journals (Sweden)

    Dombrowski James E

    2011-03-01

    Full Text Available Abstract Background Choke, caused by the endophytic fungus Epichloë typhina, is an important disease affecting orchardgrass (Dactylis glomerata L. seed production in the Willamette Valley. Little is known concerning the conditions necessary for successful infection of orchardgrass by E. typhina. Detection of E. typhina in plants early in the disease cycle can be difficult due to the sparse distribution of hyphae in the plant. Therefore, a sensitive method to detect fungal infection in plants would provide an invaluable tool for elucidating the conditions for establishment of infection in orchardgrass. Utilization of a marker gene, such as the green fluorescent protein (GFP, transformed into Epichloë will facilitate characterization of the initial stages of infection and establishment of the fungus in plants. Findings We have developed a rapid, efficient, and reproducible transformation method using electroporation of germinating Epichloë conidia isolated from infected plants. Conclusions The GFP labelled E. typhina provides a valuable molecular tool to researchers studying conditions and mechanisms involved in the establishment of choke disease in orchardgrass.

  16. Irreversible electroporation of human primary uveal melanoma in enucleated eyes.

    Directory of Open Access Journals (Sweden)

    Yossi Mandel

    Full Text Available Uveal melanoma (UM is the most common primary intraocular tumor in adults and is characterized by high rates of metastatic disease. Although brachytherapy is the most common globe-sparing treatment option for small- and medium-sized tumors, the treatment is associated with severe adverse reactions and does not lead to increased survival rates as compared to enucleation. The use of irreversible electroporation (IRE for tumor ablation has potential advantages in the treatment of tumors in complex organs such as the eye. Following previous theoretical work, herein we evaluate the use of IRE for uveal tumor ablation in human ex vivo eye model. Enucleated eyes of patients with uveal melanoma were treated with short electric pulses (50-100 µs, 1000-2000 V/cm using a customized electrode design. Tumor bioimpedance was measured before and after treatment and was followed by histopathological evaluation. We found that IRE caused tumor ablation characterized by cell membrane disruption while sparing the non-cellular sclera. Membrane disruption and loss of cellular capacitance were also associated with significant reduction in total tumor impedance and loss of impedance frequency dependence. The effect was more pronounced near the pulsing electrodes and was dependent on time from treatment to fixation. Future studies should further evaluate the potential of IRE as an alternative method of uveal melanoma treatment.

  17. In situ formation of magnetopolymersomes via electroporation for MRI

    Science.gov (United States)

    Bain, Jennifer; Ruiz-Pérez, Lorena; Kennerley, Aneurin J.; Muench, Stephen P.; Thompson, Rebecca; Battaglia, Giuseppe; Staniland, Sarah S.

    2015-09-01

    As the development of diagnostic/therapeutic (and combined: theranostic) nanomedicine grows, smart drug-delivery vehicles become ever more critical. Currently therapies consist of drugs tethered to, or encapsulated within nanoparticles or vesicles. There is growing interest in functionalising them with magnetic nanoparticles (MNPs) to target the therapeutics by localising them using magnetic fields. An alternating magnetic field induces remote heating of the particles (hyperthermia) triggering drug release or cell death. Furthermore, MNPs are diagnostic MRI contrast agents. There is considerable interest in MNP embedded vehicles for nanomedicine, but their development is hindered by difficulties producing consistently monodisperse MNPs and their reliable loading into vesicles. Furthermore, it is highly advantageous to "trigger" MNP production and to tune the MNP's size and magnetic response. Here we present the first example of a tuneable, switchable magnetic delivery vehicle for nanomedical application. These are comprised of robust, tailored polymer vesicles (polymersomes) embedded with superparamagnetic magnetite MNPs (magnetopolymersomes) which show good MRI contrast (R2* = 148.8 s-1) and have a vacant core for loading of therapeutics. Critically, the magnetopolymersomes are produced by a pioneering nanoreactor method whereby electroporation triggers the in situ formation of MNPs within the vesicle membrane, offering a switchable, tuneable magnetic responsive theranostic delivery vehicle.

  18. Optimization of single-cell electroporation protocol for forced gene expression in primary neuronal cultures.

    Science.gov (United States)

    Nishikawa, Shin; Hirashima, Naohide; Tanaka, Masahiko

    2014-09-01

    The development and function of the central nervous system (CNS) are realized through interactions between many neurons. To investigate cellular and molecular mechanisms of the development and function of the CNS, it is thus crucial to be able to manipulate the gene expression of single neurons in a complex cell population. We recently developed a technique for gene silencing by introducing small interfering RNA into single neurons in primary CNS cultures using single-cell electroporation. However, we had not succeeded in forced gene expression by introducing expression plasmids using single-cell electroporation. In the present study, we optimized the experimental conditions to enable the forced expression of green fluorescent protein (GFP) in cultured cerebellar Purkinje neurons using single-cell electroporation. We succeeded in strong GFP expression in Purkinje neurons by increasing the inside diameter of micropipettes or by making the size of the original plasmid smaller by digestion and cyclizing it by ligation. Strong GFP expression in Purkinje neurons electroporated under the optimal conditions continued to be observed for more than 25 days after electroporation. Thus, this technique could be used for forced gene expression in single neurons to investigate cellular and molecular mechanisms of the development, function, and disease of the CNS.

  19. Efficient delivery of DNA and morpholinos into mouse preimplantation embryos by electroporation.

    Directory of Open Access Journals (Sweden)

    Hui Peng

    Full Text Available Mouse preimplantation development is characterized by three major transitions and two lineage segregations. Each transition or lineage segregation entails pronounced changes in the pattern of gene expression. Thus, research into the function of genes with obvious changes in expression pattern will shed light on the molecular basis of preimplantation development. We have described a simplified and effective method--electroporation--of introducing plasmid DNA and morpholinos into mouse preimplantation embryos and verified effectiveness of this approach by testing the procedure on the endogenous gene Oct4. Before electroporation, the zona pellucida was weakened by the treatment of acid Tyrode's solution. Then we optimized the parameters such as voltage, pulse duration, number of pulses and repeats, and applied these parameters to subsequent experiments. Compared with the control groups, the number of apoptotic cells and the expression and localization of OCT3/4 or CDX2 was not significantly changed in blastocysts developed from 1-cell embryos, which were electroporated with pIRES2-AcGFP1-Nuc eukaryotic expression vector or mismatched morpholino oligonucleotides. Furthermore, electroporated plasmid DNA and morpholinos targeting the endogenous gene Oct4 were able to sharply down regulate expression of OCT4 protein and actually cause expected phenotypes in mouse preimplantation embryos. In conclusion, plasmid DNA and morpholinos could be efficient delivered into mouse preimplantation embryos by electroporation and exert their functions, and normal development of preimplantation embryos was not affected.

  20. Seeing the electroporative uptake of cell-membrane impermeable fluorescent molecules and nanoparticles.

    Science.gov (United States)

    Kim, Kisoo; Kim, Jeong Ah; Lee, Soon-Geul; Lee, Won Gu

    2012-08-21

    This paper presents direct visualization of uptake directionality for cell-membrane impermeant fluorescent molecules and fluorescence-doped nanoparticles at a single-cell level during electroporation. To observe directly the uptake direction, we used microchannel-type electroporation that can generate a relatively symmetric and uniform electric field. For all the image frames during electroporation, fluorescence intensities that occurred at cell membranes in both uptake directions toward the electrodes have been sequentially recorded and quantitatively analyzed pixel by pixel. In our experiments, we found that fluorescent molecules, even not labeled to target biomolecules, had their own uptake direction with different intensities. It is also observed that the uptake intensity toward the cell membrane had a maximal value at a certain electric voltage, not at the highest value of voltages applied. The results also imply that the uptake direction of fluorescence-doped nanoparticles can be determined by a net surface charge of uptake materials and sizes in the electroporative environments. In summary, we performed a quantitative screening and direct visualization of uptake directionality for a set of fluorescent molecules and fluorescence-doped nanoparticles using electric-pulsation. Taking a closer look at the uptake direction of exogenous materials will help researchers to understand an unknown uptake phenomenon in which way foreign materials are inclined to move, and furthermore to design functional nanoparticles for electroporative gene delivery.

  1. Electroporative transfection with KGF-1 DNA improves wound healing in a diabetic mouse model.

    Science.gov (United States)

    Marti, G; Ferguson, M; Wang, J; Byrnes, C; Dieb, R; Qaiser, R; Bonde, P; Duncan, M D; Harmon, J W

    2004-12-01

    We recently demonstrated that electroporation enhances transfection in a mouse wound-healing model. Keratinocyte growth factor (KGF) is an inducer of epithelial cell proliferation and differentiation and has been shown to be under expressed in the wounds of diabetic individuals. We hypothesized that KGF delivered into an excisional wound via naked DNA injection with subsequent electroporation would be a novel and potentially effective method to enhance wound closure in a diabetic mouse model. ELISA assays confirmed production of KGF protein in cultured mouse cells and RT-PCR assays confirmed KGF mRNA in skin samples taken from mice. In all, 32 genetically diabetic mice were given two identical excisional wounds of their dorsum and split into two groups with one group receiving KGF DNA injection and electroporation with the other group receiving no treatment. Over 90% of wounds healed in the presence of KGF and electroporation versus 40% in the untreated group by day 12. Histological analysis of the wounds demonstrated that untreated wounds contained microulcers with thin or incomplete epithelium with unresolved inflammation as compared to treated wounds where intact and mature epithelium was observed. Taken together these findings suggest that a single injection of KGF DNA encoded on a plasmid coupled with electroporation improves and accelerates wound closure in a delayed wound-healing model.

  2. Electroporation on microchips: the harmful effects of pH changes and scaling down.

    Science.gov (United States)

    Li, Yang; Wu, Mengxi; Zhao, Deyao; Wei, Zewen; Zhong, Wenfeng; Wang, Xiaoxia; Liang, Zicai; Li, Zhihong

    2015-12-14

    Electroporation has been widely used in delivering foreign biomolecules into cells, but there is still much room for improvement, such as cell viability and integrity. In this manuscript, we investigate the distribution and the toxicity of pH changes during electroporation, which significantly decreases cell viability. A localized pH gradient forms between anode and cathode leading to a localized distribution of cell death near the electrodes, especially cathodes. The toxicity of hydroxyl ions is severe and acute due to their effect in the decomposition of phospholipid bilayer membrane. On the other hand, the electric field used for electroporation aggravates the toxicity of hydroxyl because the electropermeabilization of cell membrane makes bilayer structure more loosen and vulnerable. We also investigate the side effects during scaling down the size of electrodes in electroporation microchips. Higher percentage of cells is damaged when the size of electrodes is smaller. At last, we propose an effective strategy to constrain the change of pH by modifying the composition of electroporation buffer. The modified buffer decreases the changes of pH, thus enables high cell viability even when the electric pulse duration exceeds several milliseconds. This ability has potential advantage in some applications that require long-time electric pulse stimulation.

  3. Pharmacokinetics and penetration into synovial fluid of systemical and electroporation administered sinomenine to rabbits.

    Science.gov (United States)

    Yan, Huan; Yan, Miao; Li, Huan-De; Jiang, Pei; Deng, Yang; Cai, Hua-Lin

    2015-06-01

    Sinomenine is an anti-rheumatoid arthritis (RA) drug derived from the Sinomenium acutum. The major site of RA treatment is within the synovial compartment. However, the pharmacokinetic and penetration into synovial fluid (SF) of sinomenine have not been reported. In our study, the pharmacokinetics and penetration into SF of systemic and electroporation administered sinomenine were investigated by microdialysis incorporated with HPLC-MS/MS. Sinomenine went into plasma and SF more rapidly with higher peak concentration (Cmax ) by intramuscular injection compared with oral administration. The area under the concentration-time graph (AUC0-∞ ) of intramuscularly injected sinomenine was 1,403,294.75 ± 125,534.567 ng min/mL in plasma and 456,116.37 ± 62,648.36 ng min/mL in SF, which were equivalent with those for an oral dose. These results indicated that equal amounts of sinomenine could penetrate into SF by the two administration routes, and the permeation ratios were approximately 1:3. The AUC0-∞ and Cmax were lower with electroporation compared with systemic administration, but the CSF /CPlasma (concentration of sinomenine in SF vs that of plasma) at 90, 120, 150, 180, 240 and 480 min by electroporation was 3- to 10-fold higher relative to systemic administration. This illustrated that sinomenine can be targeted into joints by electroporation, and electroporation is a potential technique for sinomenine's transdermal delivery.

  4. Intracellular tracking of single native molecules with electroporation-delivered quantum dots.

    Science.gov (United States)

    Sun, Chen; Cao, Zhenning; Wu, Min; Lu, Chang

    2014-11-18

    Quantum dots (QDs) have found a wide range of biological applications as fluorophores due to their extraordinary brightness and high photostability that are far superior to those of conventional organic dyes. These traits are particularly appealing for studying cell biology under a cellular autofluorescence background and with a long observation period. However, it remains the most important open challenge to target QDs at native intracellular molecules and organelles in live cells. Endocytosis-based delivery methods lead to QDs encapsulated in vesicles that have their surface biorecognition element hidden from the intracellular environment. The probing of native molecules using QDs has been seriously hindered by the lack of consistent approaches for delivery of QDs with exposed surface groups. In this study, we demonstrate that electroporation (i.e., the application of short electric pulses for cell permeabilization) generates reproducible results for delivering QDs into cells. We show evidence that electroporation-based delivery does not involve endocytosis or vesicle encapsulation of QDs. The amount of QD loading and the resulting cell viability can be adjusted by varying the parameters associated with the electroporation operation. To demonstrate the application of our approach for intracellular targeting, we study single-molecule motility of kinesin in live cells by labeling native kinesins using electroporation-delivered QDs. We envision that electroporation may serve as a simple and universal tool for delivering QDs into cells to label and probe native molecules and organelles.

  5. Optimising costs in WLCG operations

    CERN Document Server

    Pradillo, Mar; Flix, Josep; Forti, Alessandra; Sciabà, Andrea

    2015-01-01

    The Worldwide LHC Computing Grid project (WLCG) provides the computing and storage resources required by the LHC collaborations to store, process and analyse the 50 Petabytes of data annually generated by the LHC. The WLCG operations are coordinated by a distributed team of managers and experts and performed by people at all participating sites and from all the experiments. Several improvements in the WLCG infrastructure have been implemented during the first long LHC shutdown to prepare for the increasing needs of the experiments during Run2 and beyond. However, constraints in funding will affect not only the computing resources but also the available effort for operations. This paper presents the results of a detailed investigation on the allocation of the effort in the different areas of WLCG operations, identifies the most important sources of inefficiency and proposes viable strategies for optimising the operational cost, taking into account the current trends in the evolution of the computing infrastruc...

  6. Optimising Comprehensibility in Interlingual Translation

    DEFF Research Database (Denmark)

    Nisbeth Jensen, Matilde

    2015-01-01

    . It is argued that Plain Language writing is a type of intralingual translation as it involves rewriting or translating a complex monolingual text into comprehensible language. Based on Plain Language literature, a comprehensibility framework is elaborated, which is subsequently exemplified through...... the functional text type of Patient Information Leaflet. Finally, the usefulness of applying the principles of Plain Language and intralingual translation for optimising comprehensibility in interlingual translation is discussed....... information on medication and tax information. Such texts are often written by experts and received by lay people, and, in today’s globalised world, they are often translated as well. In these functional texts, the receiver is not a mere recipient of information, but s/he needs to be able to act upon it...

  7. Dynamic effects of point source electroporation on the rat brain tissue.

    Science.gov (United States)

    Sharabi, Shirley; Last, David; Guez, David; Daniels, Dianne; Hjouj, Mohammad Ibrahim; Salomon, Sharona; Maor, Elad; Mardor, Yael

    2014-10-01

    In spite of aggressive therapy, existing treatments offer poor prognosis for glioblastoma multiforme due to tumor infiltration into the surrounding brain as well as poor blood-brain barrier penetration of most therapeutic agents. In this paper we present a novel approach for a minimally invasive treatment and a non-invasive response assessment methodology consisting of applying intracranial point-source electroporation and assessing treatment effect volumes using magnetic resonance imaging. Using a unique setup of a single intracranial electrode and an external surface electrode we treated rats' brains with various electroporation protocols and applied magnetic resonance imaging to study the dependence of the physiological effects on electroporation treatment parameters. The extent of blood-brain barrier disruption and later volumes of permanent brain tissue damage were found to correlate significantly with the treatment voltages (r(2)=0.99, pelectroporation when planning a treatment for brain tumors.

  8. Lightning-triggered electroporation and electrofusion as possible contributors to natural horizontal gene transfer.

    Science.gov (United States)

    Kotnik, Tadej

    2013-09-01

    Phylogenetic studies show that horizontal gene transfer (HGT) is a significant contributor to genetic variability of prokaryotes, and was perhaps even more abundant during the early evolution. Hitherto, research of natural HGT has mainly focused on three mechanisms of DNA transfer: conjugation, natural competence, and viral transduction. This paper discusses the feasibility of a fourth such mechanism--cell electroporation and/or electrofusion triggered by atmospheric electrostatic discharges (lightnings). A description of electroporation as a phenomenon is followed by a review of experimental evidence that electroporation of prokaryotes in aqueous environments can result in release of non-denatured DNA, as well as uptake of DNA from the surroundings and transformation. Similarly, a description of electrofusion is followed by a review of experiments showing that prokaryotes devoid of cell wall can electrofuse into hybrids expressing the genes of their both precursors. Under sufficiently fine-tuned conditions, electroporation and electrofusion are efficient tools for artificial transformation and hybridization, respectively, but the quantitative analysis developed here shows that conditions for electroporation-based DNA release, DNA uptake and transformation, as well as for electrofusion are also present in many natural aqueous environments exposed to lightnings. Electroporation is thus a plausible contributor to natural HGT among prokaryotes, and could have been particularly important during the early evolution, when the other mechanisms might have been scarcer or nonexistent. In modern prokaryotes, natural absence of the cell wall is rare, but it is reasonable to assume that the wall has formed during a certain stage of evolution, and at least prior to this, electrofusion could also have contributed to natural HGT. The concluding section outlines several guidelines for assessment of the feasibility of lightning-triggered HGT.

  9. Immunologic response to tumor ablation with irreversible electroporation.

    Directory of Open Access Journals (Sweden)

    Xiaoxiang Li

    Full Text Available BACKGROUND: Irreversible electroporation (IRE is a promising technique for the focal treatment of pathologic tissues, which involves placing minimally invasive electrodes within the targeted region. However, the knowledge about the therapeutic efficacy and immune reactions in response to IRE remains in its infancy. METHODS: In this work, to detect whether tumor ablation with IRE could trigger the immunologic response, we developed an osteosarcoma rat model and applied IRE directly to ablate the tumor. In the experiment, 118 SD rats were randomized into 4 groups: the control, sham operation, surgical resection, and IRE groups. Another 28 rats without tumor cell implantation served as the normal non-tumor-bearing group. We analyzed the changes in T lymphocyte subsets, sIL-2R and IL-10 levels in the peripheral blood one day before operation, as well as at 1, 3, 7,14 and 21 days after the operation. Moreover, splenocytes were assayed for IFN-γ and IL-4 production using intracellular cytokine staining one day before the operation, as well as at 7 and 21 days after operation. RESULTS: We found that direct IRE completely ablated the tumor cells. A significant increase in peripheral lymphocytes, especially CD3(+ and CD4(+ cells, as well as an increased ratio of CD4(+/CD8(+ were detectable 7 days after operation in both the IRE and surgical resection groups. Compared with the surgical resection group, the IRE group exhibited a stronger cellular immune response. The sIL-2R level of the peripheral blood in the IRE group decreased with time and was significantly different from that in the surgical resection group. Moreover, ablation with IRE significantly increased the percentage of IFN-γ-positive splenocytes. CONCLUSION: These findings indicated that IRE could not only locally destroy the tumor but also change the status of cellular immunity in osteosarcoma-bearing rats. This provides experimental evidence for the clinical application of IRE in

  10. Detection of electroporation-induced membrane permeabilization states in the brain using diffusion-weighted MRI

    DEFF Research Database (Denmark)

    Mahmood, Faisal; Hansen, Rasmus H; Agerholm-Larsen, Birgit

    2015-01-01

    BACKGROUND: Tissue permeabilization by electroporation (EP) is a promising technique to treat certain cancers. Non-invasive methods for verification of induced permeabilization are important, especially in deep-seated cancers. In this study we evaluated diffusion-weighted magnetic resonance imaging...... (DW-MRI) as a quantitative method for detecting EP-induced membrane permeabilization of brain tissue using a rat brain model. MATERIAL AND METHODS: Fifty-four anesthetized Sprague-Dawley male rats were electroporated in the right hemisphere, using different voltage levels to induce no permeabilization......-induced permeabilization of brain tissue and to some extent of differentiating NP, TMP and PMP using appropriate scan timing....

  11. Clinical development of intramuscular electroporation: providing a "boost" for DNA vaccines.

    Science.gov (United States)

    Khan, Amir S; Broderick, Kate E; Sardesai, Niranjan Y

    2014-01-01

    The development of effective vaccines has helped to eradicate or control the spread of numerous infectious diseases. However, there are many more diseases that have proved more difficult to eliminate using conventional vaccines. The recent innovation of DNA vaccines may provide a "boost" to the development efforts. While the early efforts of DNA vaccines in the clinic were disappointing, the use of in vivo electroporation has helped to provide some basis for optimism. Now, there are several ongoing clinical studies of vaccines against such diseases as malaria, HIV, hepatitis C, and even various types of cancer. This review will highlight three recently published clinical studies using intramuscular DNA administration with electroporation.

  12. Theoretical and experimental study of electroporation of red blood cells using MEMS technology

    KAUST Repository

    Deng, Peigang

    2010-01-01

    A theoretical and experimental study of electroporation (EP) of red blood cells (RBCs) was presented in this paper. With additional strain energy, an energy-based model of an electropore induced on a RBC\\'s membrane at different electric fields was proposed to predict the critical EP electric field strength. In addition, EP experiments with red blood cells at single-cell level was carried out on a micro EP chip. The measured critical EP electric field strengths are in agreement with the numerical predictions. ©2010 IEEE.

  13. Systematic delay-driven power optimisation and power-driven delay optimisation of combinational circuits

    OpenAIRE

    Mehrotra, Rashmi

    2013-01-01

    With the proliferation of mobile wireless communication and embedded systems, the energy efficiency becomes a major design constraint. The dissipated energy is often referred as the product of power dissipation and the input-output delay. Most of electronic design automation techniques focus on optimising only one of these parameters either power or delay. Industry standard design flows integrate systematic methods of optimising either area or timing while for power consumption optimisation o...

  14. Benchmarks for dynamic multi-objective optimisation

    CSIR Research Space (South Africa)

    Helbig, M

    2013-06-01

    Full Text Available When algorithms solve dynamic multi-objective optimisation problems (DMOOPs), benchmark functions should be used to determine whether the algorithm can overcome specific difficulties that can occur in real-world problems. However, for dynamic multi...

  15. Topology optimisation for natural convection problems

    CERN Document Server

    Alexandersen, Joe; Andreasen, Casper Schousboe; Sigmund, Ole

    2014-01-01

    This paper demonstrates the application of the density-based topology optimisation approach for the design of heat sinks and micropumps based on natural convection effects. The problems are modelled under the assumptions of steady-state laminar flow using the incompressible Navier-Stokes equations coupled to the convection-diffusion equation through the Boussinesq approximation. In order to facilitate topology optimisation, the Brinkman approach is taken to penalise velocities inside the solid domain and the effective thermal conductivity is interpolated in order to accommodate differences in thermal conductivity of the solid and fluid phases. The governing equations are discretised using stabilised finite elements and topology optimisation is performed for two different problems using discrete adjoint sensitivity analysis. The study shows that topology optimisation is a viable approach for designing heat sink geometries cooled by natural convection and micropumps powered by natural convection.

  16. Topology Optimisation for Coupled Convection Problems

    DEFF Research Database (Denmark)

    Alexandersen, Joe

    This thesis deals with topology optimisation for coupled convection problems. The aim is to extend and apply topology optimisation to steady-state conjugate heat transfer problems, where the heat conduction equation governs the heat transfer in a solid and is coupled to thermal transport...... in a surrounding uid, governed by a convection-diffusion equation, where the convective velocity field is found from solving the isothermal incompressible steady-state Navier-Stokes equations. Topology optimisation is also applied to steady-state natural convection problems. The modelling is done using stabilised...... finite elements, the formulation and implementation of which was done partly during a special course as prepatory work for this thesis. The formulation is extended with a Brinkman friction term in order to facilitate the topology optimisation of fluid flow and convective cooling problems. The derived...

  17. An optimisation method for complex product design

    Science.gov (United States)

    Li, Ni; Yi, Wenqing; Bi, Zhuming; Kong, Haipeng; Gong, Guanghong

    2013-11-01

    Designing a complex product such as an aircraft usually requires both qualitative and quantitative data and reasoning. To assist the design process, a critical issue is how to represent qualitative data and utilise it in the optimisation. In this study, a new method is proposed for the optimal design of complex products: to make the full use of available data, information and knowledge, qualitative reasoning is integrated into the optimisation process. The transformation and fusion of qualitative and qualitative data are achieved via the fuzzy sets theory and a cloud model. To shorten the design process, parallel computing is implemented to solve the formulated optimisation problems. A parallel adaptive hybrid algorithm (PAHA) has been proposed. The performance of the new algorithm has been verified by a comparison with the results from PAHA and two other existing algorithms. Further, PAHA has been applied to determine the shape parameters of an aircraft model for aerodynamic optimisation purpose.

  18. User perspectives in public transport timetable optimisation

    DEFF Research Database (Denmark)

    Jensen, Jens Parbo; Nielsen, Otto Anker; Prato, Carlo Giacomo

    2014-01-01

    The present paper deals with timetable optimisation from the perspective of minimising the waiting time experienced by passengers when transferring either to or from a bus. Due to its inherent complexity, this bi-level minimisation problem is extremely difficult to solve mathematically, since...... on the large-scale public transport network in Denmark. The timetable optimisation approach yielded a yearly reduction in weighted waiting time equivalent to approximately 45 million Danish kroner (9 million USD)....

  19. Analysing bone regeneration using topological optimisation

    Directory of Open Access Journals (Sweden)

    Diego Alexander Garzón Alvarado

    2010-04-01

    Full Text Available The present article's object is to present the mathematical foundations of topological optimisation aimed at carrying out a study of bone regeneration. Bone structure can be economically adopted to different mechanical demands responding to topological optimisation models (having "minimum" mass and "high" resistance. Such analysis is essential for formulating physical therapy in patients needing partial or total strengthening of a particular bone's tissue structure. A mathematical model is formulated, as are the methods for resolving it.

  20. Optimising costs in WLCG operations

    Science.gov (United States)

    Alandes Pradillo, María; Dimou, Maria; Flix, Josep; Forti, Alessandra; Sciabà, Andrea

    2015-12-01

    The Worldwide LHC Computing Grid project (WLCG) provides the computing and storage resources required by the LHC collaborations to store, process and analyse the 50 Petabytes of data annually generated by the LHC. The WLCG operations are coordinated by a distributed team of managers and experts and performed by people at all participating sites and from all the experiments. Several improvements in the WLCG infrastructure have been implemented during the first long LHC shutdown to prepare for the increasing needs of the experiments during Run2 and beyond. However, constraints in funding will affect not only the computing resources but also the available effort for operations. This paper presents the results of a detailed investigation on the allocation of the effort in the different areas of WLCG operations, identifies the most important sources of inefficiency and proposes viable strategies for optimising the operational cost, taking into account the current trends in the evolution of the computing infrastructure and the computing models of the experiments.

  1. OPTIMIZATION OF ELECTROPORATION PARAMETERS FOR TRANSFECTION OF PLASMID DNA INTO MURINE BONE MARROW-DERIVED DENDRITIC CELL

    Institute of Scientific and Technical Information of China (English)

    KE Shan; CHEN Xue-hua; LI Hao; LI Jian-fang; GU Qin-long; ZHU Zheng-gang; LIU Bing-ya

    2006-01-01

    Objective To explore the optimal electroporation parameters for transfection of plasmid DNA into murine bone marrow-derived dendritic cells. Methods Murine bone marrow-derived dendritic cells (DCs) were electroporated with plasmid DNA in varied conditions, such as electrical voltage, pulse time,pre-electroporation cell condition and serum concentration in electrical buffer. Inverted fluorescence microscope and flow cytometer were used to determine the transfection efficiency. Some of the DCs genetically modified under different conditions were stained with trypan-blue and its viability was observed microscopically 48h after electroporation. Results Highest transfection efficiency (22.10%) could be reached when electrical voltage was 250V and pulse time was 20ms. Refreshing the culture medium pre-electroporation may help the cells recover more easily from gene transfer.Besides, electrical buffer containing serum may benefit the viability of DC after electroporation and temperature may has little influence on transfection efficiency. Conclusion Our observations demonstrated plasmid DNA could be efficiently transferred into murine bone marrow-derived DCs by electroporation. These data may helpful for cancer research related to murine DC transfection.

  2. Approaches and challenges to optimising primary care teams’ electronic health record usage

    Directory of Open Access Journals (Sweden)

    Nancy Pandhi

    2014-07-01

    Full Text Available Background Although the presence of an electronic health record (EHR alone does not ensure high quality, efficient care, few studies have focused on the work of those charged with optimising use of existing EHR functionality.Objective To examine the approaches used and challenges perceived by analysts supporting the optimisation of primary care teams’ EHR use at a large U.S. academic health care system.Methods A qualitative study was conducted. Optimisation analysts and their supervisor were interviewed and data were analysed for themes.Results Analysts needed to reconcile the tension created by organisational mandates focused on the standardisation of EHR processes with the primary care teams’ demand for EHR customisation. They gained an understanding of health information technology (HIT leadership’s and primary care team’s goals through attending meetings, reading meeting minutes and visiting with clinical teams. Within what was organisationally possible, EHR education could then be tailored to fit team needs. Major challenges were related to organisational attempts to standardise EHR use despite varied clinic contexts, personnel readiness and technical issues with the EHR platform. Forcing standardisation upon clinical needs that current EHR functionality could not satisfy was difficult.Conclusions Dedicated optimisation analysts can add value to health systems through playing a mediating role between HIT leadership and care teams. Our findings imply that EHR optimisation should be performed with an in-depth understanding of the workflow, cognitive and interactional activities in primary care.

  3. Conducting nanosponge electroporation for affordable and high-efficiency disinfection of bacteria and viruses in water.

    Science.gov (United States)

    Liu, Chong; Xie, Xing; Zhao, Wenting; Liu, Nian; Maraccini, Peter A; Sassoubre, Lauren M; Boehm, Alexandria B; Cui, Yi

    2013-09-11

    High-efficiency, affordable, and low energy water disinfection methods are in great need to prevent diarrheal illness, which is one of the top five leading causes of death over the world. Traditional water disinfection methods have drawbacks including carcinogenic disinfection byproducts formation, energy and time intensiveness, and pathogen recovery. Here, we report an innovative method that achieves high-efficiency water disinfection by introducing nanomaterial-assisted electroporation implemented by a conducting nanosponge filtration device. The use of one-dimensional (1D) nanomaterials allows electroporation to occur at only several volts, which is 2 to 3 orders of magnitude lower than that in traditional electroporation applications. The disinfection mechanism of electroporation prevents harmful byproduct formation and ensures a fast treatment speed of 15,000 L/(h·m(2)), which is equal to a contact time of 1 s. The conducting nanosponge made from low-cost polyurethane sponge coated with carbon nanotubes and silver nanowires ensures the device's affordability. This method achieves more than 6 log (99.9999%) removal of four model bacteria, including Escherichia coli, Salmonella enterica Typhimirium, Enterococcus faecalis, and Bacillus subtilis, and more than 2 log (99%) removal of one model virus, bacteriophage MS2, with a low energy consumption of only 100 J/L.

  4. Electroporation for drug and gene delivery in the clinic: doctors go electric

    DEFF Research Database (Denmark)

    Gehl, J.

    2008-01-01

    Electroporation is a unique system for drug and gene delivery, as it is possible to very specifically target certain tissues within the body with whatever drug, gene, isotope, or other product is desired in a specific situation. An increasing number of clinical trials are being launched, and soph...

  5. High-Voltage Electroporation of Bacteria: Genetic Transformation of Campylobacter jejuni with Plasmid DNA

    Science.gov (United States)

    Miller, Jeff F.; Dower, William J.; Tompkins, Lucy S.

    1988-02-01

    Electroporation permits the uptake of DNA by mammalian cells and plant protoplasts because it induces transient permeability of the cell membrane. We investigated the utility of high-voltage electroporation as a method for genetic transformation of intact bacterial cells by using the enteric pathogen Campylobacter jejuni as a model system. This report demonstrates that the application of high-voltage discharges to bacterial cells permits genetic transformation. Our method involves exposure of a Campylobacter cell suspension to a high-voltage exponential decay discharge (5-13 kV/cm) for a brief period of time (resistance-capacitance time constant = 2.4-26 msec) in the presence of plasmid DNA. Electrical transformation of C. jejuni results in frequencies as high as 1.2 × 106 transformants per μ g of DNA. We have investigated the effects of pulse amplitude and duration, cell growth conditions, divalent cations, and DNA concentration on the efficiency of transformation. Transformants of C. jejuni obtained by electroporation contained structurally intact plasmid molecules. In addition, evidence is presented that indicates that C. jejuni possesses DNA restriction and modification systems. The use of electroporation as a method for transforming other bacterial species and guidelines for its implementation are also discussed.

  6. Magnetic resonance electrical impedance tomography for measuring electrical conductivity during electroporation.

    Science.gov (United States)

    Kranjc, M; Bajd, F; Serša, I; Miklavčič, D

    2014-06-01

    The electroporation effect on tissue can be assessed by measurement of electrical properties of the tissue undergoing electroporation. The most prominent techniques for measuring electrical properties of electroporated tissues have been voltage-current measurement of applied pulses and electrical impedance tomography (EIT). However, the electrical conductivity of tissue assessed by means of voltage-current measurement was lacking in information on tissue heterogeneity, while EIT requires numerous additional electrodes and produces results with low spatial resolution and high noise. Magnetic resonance EIT (MREIT) is similar to EIT, as it is also used for reconstruction of conductivity images, though voltage and current measurements are not limited to the boundaries in MREIT, hence it yields conductivity images with better spatial resolution. The aim of this study was to investigate and demonstrate the feasibility of the MREIT technique for assessment of conductivity images of tissues undergoing electroporation. Two objects were investigated: agar phantoms and ex vivo liver tissue. As expected, no significant change of electrical conductivity was detected in agar phantoms exposed to pulses of all used amplitudes, while a considerable increase of conductivity was measured in liver tissue exposed to pulses of different amplitudes.

  7. Improvement of in vivo transfer of plasmid DNA in muscle : Comparison of electroporation versus ultrasound

    NARCIS (Netherlands)

    Kusumanto, Yoka H.; Mulder, Nanno H.; Dam, Wendy A.; Losen, Mario H.; Meijer, Coby; Hospers, Geke A. P.

    2007-01-01

    Plasmid-based gene delivery to muscle is a treatment strategy for many diseases with potential advantages above viral-based gene delivery methods, however, with a relative low transfection efficiency. We compared two physical methods-electroporation and ultrasound-that facilitate DNA uptake into cel

  8. Electroporation ablation: A new energy modality for ablation of arrhythmogenic cardiac substrate

    NARCIS (Netherlands)

    van Driel, VJHM

    2016-01-01

    At the very end of the Direct Current (DC) era, low-energy DC ablation was demonstrated to cause myocardial lesions by non-thermal irreversible electroporation (IRE) (permanent formation of pores in the cell membrane, leading to cell death), without arcing and/or barotrauma. To eliminate rather smal

  9. A Parametric Study Delineating Irreversible Electroporation from Thermal Damage Based on a Minimally Invasive Intracranial Procedure

    Directory of Open Access Journals (Sweden)

    Ellis Thomas L

    2011-04-01

    Full Text Available Abstract Background Irreversible electroporation (IRE is a new minimally invasive technique to kill undesirable tissue in a non-thermal manner. In order to maximize the benefits from an IRE procedure, the pulse parameters and electrode configuration must be optimized to achieve complete coverage of the targeted tissue while preventing thermal damage due to excessive Joule heating. Methods We developed numerical simulations of typical protocols based on a previously published computed tomographic (CT guided in vivo procedure. These models were adapted to assess the effects of temperature, electroporation, pulse duration, and repetition rate on the volumes of tissue undergoing IRE alone or in superposition with thermal damage. Results Nine different combinations of voltage and pulse frequency were investigated, five of which resulted in IRE alone while four produced IRE in superposition with thermal damage. Conclusions The parametric study evaluated the influence of pulse frequency and applied voltage on treatment volumes, and refined a proposed method to delineate IRE from thermal damage. We confirm that determining an IRE treatment protocol requires incorporating all the physical effects of electroporation, and that these effects may have significant implications in treatment planning and outcome assessment. The goal of the manuscript is to provide the reader with the numerical methods to assess multiple-pulse electroporation treatment protocols in order to isolate IRE from thermal damage and capitalize on the benefits of a non-thermal mode of tissue ablation.

  10. Regional electroporation of single cardiac myocytes in a focused electric field.

    Science.gov (United States)

    Klauke, Norbert; Smith, Godfrey; Cooper, Jonathan M

    2010-01-15

    There is now a significant interest in being able to locate single cells within geometrically defined regions of a microfluidic chip and to gain intracellular access through the local electroporation of the cell membrane. This paper describes the microfabrication of electroporation devices which can enable the regional electroporation of adult ventricular myocytes, in order to lower the local electrical resistance of the cell membrane. Initially three different devices, designed to suit the characteristic geometry of the cardiomyocyte, were investigated (all three designs serve to focus the electric field to selected regions of the cell). We demonstrate that one of these three devices revealed the sequence of cellular responses to field strengths of increasing magnitudes, namely, cell contraction, hypercontraction, and lysis. This same device required a reduced threshold voltage for each of these events, including in particular membrane breakdown. We were not only able to show the gradual regional increase in the electric conductivity of the cell membrane but were also able to avoid changes in the local intra- and extracellular pH (by preventing the local generation of protons at the electrode surface, as a consequence of the reduced threshold voltage). The paper provides evidence for new strategies for achieving robust and reproducible regional electroporation, a technique which, in future, may be used for the insertion of large molecular weight molecules (including genes) as well as for on-chip voltage clamping of the primary adult cardiomyocyte.

  11. Injection molded chips with integrated conducting polymer electrodes for electroporation of cells

    DEFF Research Database (Denmark)

    Andresen, Kristian; Hansen, Morten; Matschuk, Maria

    2010-01-01

    We present the design-concept for an all polymer injection molded single use microfluidic device. The fabricated devices comprise integrated conducting polymer electrodes and Luer fitting ports to allow for liquid and electrical access. A case study of low voltage electroporation of biological ce...

  12. A new principle of cell sorting by using selective electroporation in a modified flow cytometer

    NARCIS (Netherlands)

    Bakker Schut, Tom C.; Grooth, de Bart G.; Greve, Jan

    1990-01-01

    When a strong electric field pulse of a few microseconds is applied to biological cells, small pores are formed in the cell membranes; this process is called electroporation. At high field strengths and/or long pulse durations the membranes will be damaged permanently. This eventually leads to cell

  13. Loading of acute myeloid leukemia cells with poly(I:C) by electroporation

    NARCIS (Netherlands)

    Lion, E.; Winde, C.M. de; Tendeloo, V.F. Van; Smits, E.L.

    2014-01-01

    In this chapter, we describe the technique of electroporation as an efficient method to load primary leukemic cells with the double-stranded RNA (dsRNA) analogue, polyriboinosinic polyribocytidylic acid (poly(I:C)), and detail on the delicate freezing and thawing procedure of primary leukemic cells.

  14. Irreversible electroporation ablation area enhanced by synergistic high- and low-voltage pulses

    Science.gov (United States)

    2017-01-01

    Irreversible electroporation (IRE) produced by a pulsed electric field can ablate tissue. In this study, we achieved an enhancement in ablation area by using a combination of short high-voltage pulses (HVPs) to create a large electroporated area and long low-voltage pulses (LVPs) to ablate the electroporated area. The experiments were conducted in potato tuber slices. Slices were ablated with an array of four pairs of parallel steel electrodes using one of the following four electric pulse protocols: HVP, LVP, synergistic HVP+LVP (SHLVP) or LVP+HVP. Our results showed that the SHLVPs more effectively necrotized tissue than either the HVPs or LVPs, even when the SHLVP dose was the same as or lower than the HVP or LVP doses. The HVP and LVP order mattered and only HVPs+LVPs (SHLVPs) treatments increased the size of the ablation zone because the HVPs created a large electroporated area that was more susceptible to the subsequent LVPs. Real-time temperature change monitoring confirmed that the tissue was non-thermally ablated by the electric pulses. Theoretical calculations of the synergistic effects of the SHLVPs on tissue ablation were performed. Our proposed SHLVP protocol provides options for tissue ablation and may be applied to optimize the current clinical IRE protocols. PMID:28253331

  15. Doxorubicin delivery enhanced by electroporation to gastrointestinal adenocarcinoma cells with P-gp overexpression.

    Science.gov (United States)

    Kulbacka, Julita; Daczewska, Małgorzata; Dubińska-Magiera, Magda; Choromańska, Anna; Rembiałkowska, Nina; Surowiak, Paweł; Kulbacki, Marek; Kotulska, Małgorzata; Saczko, Jolanta

    2014-12-01

    Electroporation (EP) can effectively support the penetration of macromolecules from the extracellular space into cells. Electropores induced by the influence of electromagnetic field generate additional paths of transport for macromolecules. The aim of this study was evaluation of the electroporation effect on doxorubicin transport efficiency to human colon (LoVo and LoVo/DX) and gastric (EPG85-257/P and EPG85-257/RDB) adenocarcinoma cells with overexpression of P-glycoprotein and murine macrophage cell line (P388/D1). In our EP experiments cells were placed into a cuvette with aluminum electrodes and pulsed with five square electric pulses of 1300 V/cm and duration of 50 μs each. Cells were also treated with low doxorubicin concentration ([DOX]=1.7 μM). The ultrastructure (TEM) and changes of P-glycoprotein expression of tumor cells subjected to electric field were monitored. The mitochondrial cell function and trypan blue staining were evaluated after 24h. Our results indicate the most pronounced effect of EP with DOX and disturbed ultrastructure in resistant gastric and colon cells with decrease of P-gp expression. Electroporation may be an attractive delivery method of cytostatic drugs in chemotherapy, enabling reduction of drug dose, exposure time and side effects.

  16. Optimizing clinical performance and geometrical robustness of a new electrode device for intracranial tumor electroporation

    DEFF Research Database (Denmark)

    Mahmood, Faisal; Gehl, Julie

    2011-01-01

    Current technology has limited applicability for electroporation based treatment of deep-seated tumors, and is in general, not optimized in terms of compliance with clinically relevant parameters. Here we present a novel electrode device developed for electrotransfer of antineoplastic drugs...

  17. Transformations of the gram-positive honey bee pathogen, Paenibacillus larvae, by electroporation

    Science.gov (United States)

    In this study we developed an electrotransformation method for use with the Gram-positive bacterium Paenibacillus larvae—a deadly pathogen of honey bees. The method is substantially different from the only other electroporation method for a Paenibacillus species found in the literature. Using the ty...

  18. Nanowire-Modified Three-Dimensional Electrode Enabling Low-Voltage Electroporation for Water Disinfection.

    Science.gov (United States)

    Huo, Zheng-Yang; Xie, Xing; Yu, Tong; Lu, Yun; Feng, Chao; Hu, Hong-Ying

    2016-07-19

    More than 10% of the people in the world still suffer from inadequate access to clean water. Traditional water disinfection methods (e.g., chlorination and ultraviolet radiation) include concerns about the formation of carcinogenic disinfection byproducts (DBPs), pathogen reactivation, and/or excessive energy consumption. Recently, a nanowire-assisted electroporation-disinfection method was introduced as an alternative. Here, we develop a new copper oxide nanowire (CuONW)-modified three-dimensional copper foam electrode using a facile thermal oxidation approach. An electroporation-disinfection cell (EDC) equipped with two such electrodes has achieved superior disinfection performance (>7 log removal and no detectable bacteria in the effluent). The disinfection mechanism of electroporation guarantees an exceedingly low operation voltage (1 V) and level of energy consumption (25 J L(-1)) with a short contact time (7 s). The low operation voltage avoids chlorine generation and thus reduces the potential of DBP formation. Because of irreversible electroporation damage on cell membranes, no regrowth and/or reactivation of bacteria occurs during storage after EDC treatment. Water disinfection using EDCs has great potential for practical applications.

  19. Electroporation-induced siRNA precipitation obscures the efficiency of siRNA loading into extracellular vesicles.

    Science.gov (United States)

    Kooijmans, Sander A A; Stremersch, Stephan; Braeckmans, Kevin; de Smedt, Stefaan C; Hendrix, An; Wood, Matthew J A; Schiffelers, Raymond M; Raemdonck, Koen; Vader, Pieter

    2013-11-28

    Extracellular vesicles (EVs) are specialised endogenous carriers of proteins and nucleic acids and are involved in intercellular communication. EVs are therefore proposed as candidate drug delivery systems for the delivery of nucleic acids and other macromolecules. However, the preparation of EV-based drug delivery systems is hampered by the lack of techniques to load the vesicles with nucleic acids. In this work we have now characterised in detail the use of an electroporation method for this purpose. When EVs were electroporated with fluorescently labelled siRNA, siRNA retention was comparable with previously published results (20-25% based on fluorescence spectroscopy and fluorescence fluctuation spectroscopy), and electroporation with unlabelled siRNA resulted in significant siRNA retention in the EV pellet as measured by RT-PCR. Remarkably, when siRNA was electroporated in the absence of EVs, a similar or even greater siRNA retention was measured. Nanoparticle tracking analysis and confocal microscopy showed extensive formation of insoluble siRNA aggregates after electroporation, which could be dramatically reduced by addition of EDTA. Other strategies to reduce aggregate formation, including the use of cuvettes with conductive polymer electrodes and the use of an acidic citrate electroporation buffer, resulted in a more efficient reduction of siRNA precipitation than EDTA. However, under these conditions, siRNA retention was below 0.05% and no significant differences in siRNA retention could be measured between samples electroporated in the presence or absence of EVs. Our results show that electroporation of EVs with siRNA is accompanied by extensive siRNA aggregate formation, which may cause overestimation of the amount of siRNA actually loaded into EVs. Moreover, our data clearly illustrate that electroporation is far less efficient than previously described, and highlight the necessity for alternative methods to prepare siRNA-loaded EVs.

  20. Use of electroporation and reverse iontophoresis for extraction of transdermal multibiomarkers

    Directory of Open Access Journals (Sweden)

    Ching CTS

    2012-02-01

    Full Text Available Congo Tak-Shing Ching1,2, Lin-Shien Fu3-5, Tai-Ping Sun1, Tzu-Hsiang Hsu1, Kang-Ming Chang21Department of Electrical Engineering, National Chi Nan University, Puli, Nantou County, 2Department of Photonics and Communication Engineering, Asia University, Wufeng, Taichung, 3Department of Pediatrics, National Yang Ming University, Taipei, 4Institute of Technology, National Chi Nan University, Puli, 5Department of Pediatrics, Taichung Veterans General Hospital, Taichung City, TaiwanBackground: Monitoring of biomarkers, like urea, prostate-specific antigen (PSA, and osteopontin, is very important because they are related to kidney disease, prostate cancer, and ovarian cancer, respectively. It is well known that reverse iontophoresis can enhance transdermal extraction of small molecules, and even large molecules if reverse iontophoresis is used together with electroporation. Electroporation is the use of a high-voltage electrical pulse to create nanochannels within the stratum corneum, temporarily and reversibly. Reverse iontophoresis is the use of a small current to facilitate both charged and uncharged molecule transportation across the skin. The objectives of this in vitro study were to determine whether PSA and osteopontin are extractable transdermally and noninvasively and whether urea, PSA, and osteopontin can be extracted simultaneously by electroporation and reverse iontophoresis.Methods: All in vitro experiments were conducted using a diffusion cell assembled with the stratum corneum of porcine skin. Three different symmetrical biphasic direct currents (SBdc, five various electroporations, and a combination of the two techniques were applied to the diffusion cell via Ag/AgCl electrodes. The three different SBdc had the same current density of 0.3 mA/cm2, but different phase durations of 0 (ie, no current, control group, 30, and 180 seconds. The five different electroporations had the same pulse width of 1 msec and number of pulses per second

  1. Enhancement of melphalan activity by buthionine sulfoximine and electroporation in melanoma cells.

    Science.gov (United States)

    Ongaro, Alessia; Pellati, Agnese; De Mattei, Monica; De Terlizzi, Francesca; Rossi, Carlo R; Campana, Luca G

    2015-03-01

    Melphalan represents the reference drug for locoregional chemotherapy of melanoma; nevertheless, treatment failure may occur because of resistance to chemotherapy. Refractory melanoma cells show either an increased capability of drug inactivation, which is known to be associated with elevated intracellular levels of glutathione (GSH), or a decreased melphalan uptake. The aim of this study was to explore a biochemical and a biophysical strategy, and their combination, to overcome melphalan resistance in melanoma cells. The biochemical strategy was based on the treatment of melanoma cells with DL-buthionine (S,R)-sulfoximine (BSO) to deplete the GSH levels, thus reducing melphalan inactivation. In the biophysical strategy, cell membrane electroporation was used to increase melphalan uptake. The SK-MEL 28-resistant human melanoma cell line was pretreated with 50 μmol/l BSO for 24 h and then treated with increasing melphalan doses, with or without electroporation. Spectrophotometric quantification of cell viability was used to determine melphalan cytotoxicity. Intracellular total GSH was measured using a kinetic enzymatic assay. BSO induced 3.50-fold GSH depletion in untreated cells and a similar reduction was also maintained in melphalan-treated cells. BSO pretreatment produced a 2.46-fold increase in melphalan cytotoxicity. Electroporation increased melphalan cytotoxicity 1.42-fold. The combination of both BSO pretreatment with melphalan plus electroporation led to a 4.40-fold increase in melphalan cytotoxicity compared with melphalan alone. Pretreatment with BSO and cell membrane permeabilization by electroporation enhanced the cytotoxic activity of melphalan in melanoma cells. Their rational combination deserves further investigation and may improve the efficacy of locoregional chemotherapy of melanoma.

  2. Optimisation Modelling of Efficiency of Enterprise Restructuring

    Directory of Open Access Journals (Sweden)

    Yefimova Hanna V.

    2014-03-01

    Full Text Available The article considers issues of optimisation of the use of resources directed at restructuring of a shipbuilding enterprise, which is the main prerequisite of its efficiency. Restructuring is considered as a process of complex and interconnected change in the structure of assets, liabilities, enterprise functions, initiated by dynamic environment, which is based on the strategic concept of its development and directed at increase of efficiency of its activity, which is expressed in the growth of cost. The task of making a decision to restructure a shipbuilding enterprise and selection of a specific restructuring project refers to optimisation tasks of prospective planning. Enterprise resources that are allocated for restructuring serve as constraints of the mathematical model. Main criteria of optimisation are maximisation of pure discounted income or minimisation of expenditures on restructuring measures. The formed optimisation model is designed for assessment of volumes of attraction of own and borrowed funds for restructuring. Imitation model ensures development of cash flows. The task solution is achieved on the basis of the complex of interrelated optimisation and imitation models and procedures on formation, selection and co-ordination of managerial decisions.

  3. Optimisation of Investment Resources at Small Enterprises

    Directory of Open Access Journals (Sweden)

    Shvets Iryna B.

    2014-03-01

    Full Text Available The goal of the article lies in the study of the process of optimisation of the structure of investment resources, development of criteria and stages of optimisation of volumes of investment resources for small enterprises by types of economic activity. The article characterises the process of transformation of investment resources into assets and liabilities of the balances of small enterprises and conducts calculation of the structure of sources of formation of investment resources in Ukraine at small enterprises by types of economic activity in 2011. On the basis of the conducted analysis of the structure of investment resources of small enterprises the article forms main groups of criteria of optimisation in the context of individual small enterprises by types of economic activity. The article offers an algorithm and step-by-step scheme of optimisation of investment resources at small enterprises in the form of a multi-stage process of management of investment resources in the context of increase of their mobility and rate of transformation of existing resources into investments. The prospect of further studies in this direction is development of a structural and logic scheme of optimisation of volumes of investment resources at small enterprises.

  4. Multicriteria Optimisation in Logistics Forwarder Activities

    Directory of Open Access Journals (Sweden)

    Tanja Poletan Jugović

    2007-05-01

    Full Text Available Logistics forwarder, as organizer and planner of coordinationand integration of all the transport and logistics chains elements,uses adequate ways and methods in the process of planningand decision-making. One of these methods, analysed inthis paper, which could be used in optimisation of transportand logistics processes and activities of logistics forwarder, isthe multicriteria optimisation method. Using that method, inthis paper is suggested model of multicriteria optimisation of logisticsforwarder activities. The suggested model of optimisationis justified in keeping with method principles of multicriteriaoptimization, which is included in operation researchmethods and it represents the process of multicriteria optimizationof variants. Among many different processes of multicriteriaoptimization, PROMETHEE (Preference Ranking OrganizationMethod for Enrichment Evaluations and Promcalc& Gaia V. 3.2., computer program of multicriteria programming,which is based on the mentioned process, were used.

  5. Topology Optimisation for Coupled Convection Problems

    DEFF Research Database (Denmark)

    Alexandersen, Joe; Andreasen, Casper Schousboe; Aage, Niels

    conduction governs in the solid parts of the design domain and couples to convection-dominated heat transfer to a surrounding fluid. Both loosely coupled and tightly coupled problems are considered. The loosely coupled problems are convection-diffusion problems, based on an advective velocity field from......The work focuses on applying topology optimisation to forced and natural convection problems in fluid dynamics and conjugate (fluid-structure) heat transfer. To the authors' knowledge, topology optimisation has not yet been applied to natural convection flow problems in the published literature...... and the current work is thus seen as contributing new results to the field. In the literature, most works on the topology optimisation of weakly coupled convection-diffusion problems focus on the temperature distribution of the fluid, but a selection of notable exceptions also focusing on the temperature...

  6. A 'suicide' CRISPR-Cas9 system to promote gene deletion and restoration by electroporation in Cryptococcus neoformans.

    Science.gov (United States)

    Wang, Yu; Wei, Dongsheng; Zhu, Xiangyang; Pan, Jiao; Zhang, Ping; Huo, Liang; Zhu, Xudong

    2016-08-09

    Loss-of-function mutagenesis is an important tool used to characterize gene functions, and the CRISPR-Cas9 system is a powerful method for performing targeted mutagenesis in organisms that present low recombination frequencies, such as the serotype D strains of Cryptococcus neoformans. However, when the CRISPR-Cas9 system persists in the host cells, off-target effects and Cas9 cytotoxicity may occur, which might block subsequent genetic manipulation. Here, we report a method of spontaneously eliminating the CRISPR-Cas9 system without impairing its robust editing function. We successfully expressed single guide RNA under the driver of an endogenous U6 promoter and the human codon-optimized Cas9 endonuclease with an ACT1 promoter. This system can effectively generate an indel mutation and efficiently perform targeted gene disruption via homology-directed repair by electroporation in yeast. We then demonstrated the spontaneous elimination of the system via a cis arrangement of the CRISPR-Cas9 expression cassettes to the recombination construct. After a system-mediated double crossover, the CRISPR-Cas9 cassettes were cleaved and degraded, which was validated by Southern blotting. This 'suicide' CRISPR-Cas9 system enables the validation of gene functions by subsequent complementation and has the potential to minimize off-target effects. Thus, this technique has the potential for use in functional genomics studies of C. neoformans.

  7. The Effect of Electroporation of a Lyotroic Liquid Crystal Genistein-Based Formulation in the Recovery of Murine Melanoma Lesions.

    Science.gov (United States)

    Danciu, Corina; Berkó, Szilvia; Varju, Gábor; Balázs, Boglárka; Kemény, Lajos; Németh, István Balázs; Cioca, Andreea; Petruș, Alexandra; Dehelean, Cristina; Cosmin, Citu Ioan; Amaricai, Elena; Toma, Claudia Crina

    2015-07-08

    A lamellar lyotropic liquid crystal genistein-based formulation (LLC-Gen) was prepared in order to increase the aqueous solubility of the lipophilic phytocompound genistein. The formulation was applied locally, in a murine model of melanoma, with or without electroporation. The results demonstrated that, when the formulation was applied by electroporation, the tumors appeared later. During the 21 days of the experiment, the LLC-Gen formulation decreased the tumor volume, the amount of melanin and the degree of erythema, but when electroporation was applied, all these parameters indicated a better prognosis even (lower tumor volume, amount of melanin and degree of erythema). Although hematoxylin-eosin (HE) staining confirmed the above events, application of the LLC-Gen formulation by electroporation did not lead to a significant effect in terms of the serum concentrations of the protein S100B and serum neuron specific enolase (NSE), or the tissue expression of the platelet-derived growth factor receptor β (PDGFRβ) antibody.

  8. [Process optimisation: from theory to practical implementation].

    Science.gov (United States)

    Töpfer, Armin

    2010-01-01

    Today process optimisation is an indispensable approach to mastering the current challenges of modern health care management. The objective is to design business processes free of defects and free of waste as well as their monitoring and controlling with meaningful test statistics. Based on the identification of essential key performance indicators, key success factors and value cash generators two basic approaches to process optimisation, which are well-established and widely used in the industry, are now being implemented in the health care sector as well: Lean Management and Six Sigma.

  9. Simulating stem growth using topological optimisation

    Directory of Open Access Journals (Sweden)

    Carlos Alberto Narváez

    2010-04-01

    Full Text Available Engineers are currently resorting to observations of nature for making new designs. Studying the functioning of bodies of plants and animals has required them to be modelled and simulated; however, some models born from engineering problems could be used for such purposes. This article shows how topological optimisation (a mathematical model for optimising designing structural elements can be used for modeling and simulating the way a stem grows in terms of carrying out its funtion of providing support for the leaves and a plant's other upper organs.

  10. Bat Algorithm for Multi-objective Optimisation

    CERN Document Server

    Yang, Xin-She

    2012-01-01

    Engineering optimization is typically multiobjective and multidisciplinary with complex constraints, and the solution of such complex problems requires efficient optimization algorithms. Recently, Xin-She Yang proposed a bat-inspired algorithm for solving nonlinear, global optimisation problems. In this paper, we extend this algorithm to solve multiobjective optimisation problems. The proposed multiobjective bat algorithm (MOBA) is first validated against a subset of test functions, and then applied to solve multiobjective design problems such as welded beam design. Simulation results suggest that the proposed algorithm works efficiently.

  11. Topology Optimisation of Wireless Sensor Networks

    Directory of Open Access Journals (Sweden)

    Thike Aye Min

    2016-01-01

    Full Text Available Wireless sensor networks are widely used in a variety of fields including industrial environments. In case of a clustered network the location of cluster head affects the reliability of the network operation. Finding of the optimum location of the cluster head, therefore, is critical for the design of a network. This paper discusses the optimisation approach, based on the brute force algorithm, in the context of topology optimisation of a cluster structure centralised wireless sensor network. Two examples are given to verify the approach that demonstrate the implementation of the brute force algorithm to find an optimum location of the cluster head.

  12. Influence of electroporation on immunogenicity of the DNA vaccine pVAX-tG250FcGB%电穿孔技术对DNA疫苗pVAX-tG250FcGB免疫效果的影响

    Institute of Scientific and Technical Information of China (English)

    肖毅; 于继云; 高昆; 杨勇; 阎瑾琦; 张亮; 王宇; 徐元基; 田仁礼; 杜芝燕

    2013-01-01

    Objective To investigate the influence of electroporation on the immunogenicity of the DNA vaccine pVAX-tG250FcGB. Methods The DNA vaccine pVAX-tG250FcGB was constructed by inserting the coding gene of tG250 fusion genes into the expression vector pVAX. The DNA vaccine was delivered in BALB/c mouse by electroporation or intramuscular injection, and the induced antigen specific immune responses were compared. Results The vaccine delivered by electroporation and intramuscular injection both induced immune responses in BALB/c mouse, but electroporation produced an obviously stronger effect than intramuscular injection. Conclusion Electroporation-mediated DNA vaccine delivery can produce strong immune response in mice and is an effective means for studying the immunogenic effect of DNA vaccine pVAX-tG250FcGB.%目的:探讨应用电穿孔技术对DNA疫苗pVAX-tG250FcGB免疫效果的影响。方法构建肾癌DNA疫苗pVAX-tG250FcGB,通过体内电穿孔技术或普通肌肉注射途径,将DNA疫苗导入到BALB/c小鼠体内,探讨电穿孔免疫途径诱导抗原特异性免疫应答的效果。结果电穿孔技术或普通肌肉注射均能在小鼠体内诱导出抗原特异性的体液免疫应答和细胞免疫应答,电穿孔技术的免疫效果明显优于普通肌肉注射途径。结论电穿孔技术介导的DNA疫苗pVAX-tG250FcGB免疫具有较强的诱导免疫应答的能力,是研究DNA疫苗pVAX-tG250FcGB免疫效果的一种有效途径。

  13. Careful treatment planning enables safe ablation of liver tumors adjacent to major blood vessels by percutaneous irreversible electroporation (IRE

    Directory of Open Access Journals (Sweden)

    Kos Bor

    2015-09-01

    Full Text Available Background. Irreversible electroporation (IRE is a tissue ablation method, which relies on the phenomenon of electroporation. When cells are exposed to a sufficiently electric field, the plasma membrane is disrupted and cells undergo an apoptotic or necrotic cell death. Although heating effects are known IRE is considered as non-thermal ablation technique and is currently applied to treat tumors in locations where thermal ablation techniques are contraindicated.

  14. Optimisation of interventional cardiology procedures; Optimisation des procedures en cardiologie interventionnelle

    Energy Technology Data Exchange (ETDEWEB)

    Bar, Olivier [SELARL, Cardiologie Interventionnelle Imagerie Cardiaque - CIIC, 8, place de la Cathedrale - 37042 Tours (France)

    2011-07-15

    Radiation-guided procedures in interventional cardiology include diagnostic and/or therapeutic procedures, primarily coronary catheterization and coronary angioplasty. Application of the principles of radiation protection and the use of optimised procedures are contributing to dose reduction while maintaining the radiological image quality necessary for performance of the procedures. The mandatory training in patient radiation protection and technical training in the use of radiology devices mean that implementing continuous optimisation of procedures is possible in practice. This optimisation approach is the basis of patient radiation protection; when associated with the wearing of protective equipment it also contributes to the radiation protection of the cardiologists. (author)

  15. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control

    Science.gov (United States)

    Chang, Lingqian; Bertani, Paul; Gallego-Perez, Daniel; Yang, Zhaogang; Chen, Feng; Chiang, Chiling; Malkoc, Veysi; Kuang, Tairong; Gao, Keliang; Lee, L. James; Lu, Wu

    2015-12-01

    Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a ``3D nano-channel electroporation (NEP) chip'' on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40 000 cells per cm2 on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells.Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a ``3D nano-channel electroporation (NEP) chip'' on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40 000 cells per cm2 on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk

  16. 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies

    CERN Document Server

    Kramar, Peter

    2016-01-01

    This volume presents the proceedings of the 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies (WC2015). The congress took place in Portorož, Slovenia, during the week of September 6th to 10th, 2015. The scientific part of the Congress covered different aspects of electroporation and related technologies and included the following main topics:   ·         Application of pulsed electric fields technology in food: challenges and opportunities ·         Electrical impedance measurement for assessment of electroporation yield ·         Electrochemistry and electroporation ·         Electroporation meets electrostimulation ·         Electrotechnologies for food and biomass treatment ·         Food and biotechnology applications ·         In vitro electroporation - basic mechanisms ·         Interfacial behaviour of lipid-assemblies, membranes and cells in electric f...

  17. Delivery of molecules into cells using localized single cell electroporation on ITO micro-electrode based transparent chip.

    Science.gov (United States)

    Chen, Sheng-Chiech; Santra, Tuhin Subhra; Chang, Chia-Jung; Chen, Tsung-Ju; Wang, Pen-Cheng; Tseng, Fan-Gang

    2012-10-01

    Single cell electroporation is one of the nonviral method which successfully allows transfection of exogenous macromolecules into individual living cell. We present localized cell membrane electroporation at single-cell level by using indium tin oxide (ITO) based transparent micro-electrodes chip with inverted microscope. A focused ion beam (FIB) technique has been successfully deployed to fabricate transparent ITO micro-electrodes with submicron gaps, which can generate more intense electric field to produce very localized cell membrane electroporation. In our approach, we have successfully achieved 0.93 μm or smaller electroporation region on the cell surface to inject PI (Propidium Iodide) dye into the cell with 60 % cell viability. This experiments successfully demonstrate the cell self-recover process from the injected PI dye intensity variation. Our localized cell membrane electroporation technique (LSCMEP) not only generates reversible electroporation process but also it provides a clear optical path for potentially monitoring/tracking of drugs to deliver in single cell level.

  18. A flexible microneedle array as low-voltage electroporation electrodes for in vivo DNA and siRNA delivery.

    Science.gov (United States)

    Wei, Zewen; Zheng, Shuquan; Wang, Renxin; Bu, Xiangli; Ma, Huailei; Wu, Yidi; Zhu, Ling; Hu, Zhiyuan; Liang, Zicai; Li, Zhihong

    2014-10-21

    In vivo electroporation is an appealing method to deliver nucleic acid into living tissues, but the clinical application of such a method was limited due to severe tissue damage and poor coverage of the tissue surface. Here we present the validation of a novel flexible microneedle array electrode (MNAE) chip, in which the microneedle array and the flexible substrate are integrated together to simultaneously facilitate low-voltage electroporation and accomplish good coverage of the tissue surface. The efficient delivery of both DNA and siRNA was demonstrated on mice. Upon penetrating the high-resistance stratum corneum, the electroporation voltage was reduced to about 35 V, which was generally recognized safe for humans. Also, a pathological analysis of the microneedle-electroporated tissues was carried out to thoroughly assess the skin damage, which is an important consideration in pre-clinical studies of electroporation devices. This MNAE constitutes a novel way of in vivo delivery of siRNA and DNA to certain tissues or organs with satisfactory efficiency and good adaptation to the tissue surface profile as well as minimum tissue damage, thus avoiding the disadvantages of existing electroporation methods.

  19. Enhancement of therapeutic drug and DNA delivery into cells by electroporation* Enhancement of therapeutic drug and DNA delivery into cells by electroporation

    Science.gov (United States)

    Rabussay, Dietmar; Dev, Nagendu B.; Fewell, Jason; Smith, Louis C.; Widera, Georg; Zhang, Lei

    2003-02-01

    The effectiveness of potentially powerful therapeutics, including DNA, is often limited by their inability to permeate the cell membrane efficiently. Electroporation (EP) also referred to as `electropermeabilization' of the outer cell membrane renders this barrier temporarily permeable by inducing `pores' across the lipid bilayer. For in vivo EP, the drug or DNA is delivered into the interstitial space of the target tissue by conventional means, followed by local EP. EP pulses of micro- to millisecond duration and field strengths of 100-1500 V cm-1 generally enhance the delivery of certain chemotherapeutic drugs by three to four orders of magnitude and intracellular delivery of DNA several hundred-fold. We have used EP in clinical studies for human cancer therapy and in animals for gene therapy and DNA vaccination. Late stage squamous cell carcinomas of the head and neck were treated with intratumoural injection of bleomycin and subsequent EP. Of the 69 tumours treated, 25% disappeared completely and another 32% were reduced in volume by more than half. Residence time of bleomycin in electroporated tumours was significantly greater than in non-electroporated lesions. Histological findings and gene expression patterns after bleomycin-EP treatment indicated rapid apoptosis of the majority of tumour cells. In animals, we demonstrated the usefulness of EP for enhanced DNA delivery by achieving normalization of blood clotting times in haemophilic dogs, and by substantially increasing transgene expression in smooth muscle cells of arterial walls using a novel porous balloon EP catheter. Finally, we have found in animal experiments that the immune response to DNA vaccines can be dramatically enhanced and accelerated by EP and co-injection of micron-sized particles. We conclude that EP represents an effective, economical and safe approach to enhance the intracellular delivery, and thus potency, of important drugs and genes for therapeutic purposes. The safety and pharmaco

  20. Extending Particle Swarm Optimisers with Self-Organized Criticality

    DEFF Research Database (Denmark)

    Løvbjerg, Morten; Krink, Thiemo

    2002-01-01

    Particle swarm optimisers (PSOs) show potential in function optimisation, but still have room for improvement. Self-organized criticality (SOC) can help control the PSO and add diversity. Extending the PSO with SOC seems promising reaching faster convergence and better solutions.......Particle swarm optimisers (PSOs) show potential in function optimisation, but still have room for improvement. Self-organized criticality (SOC) can help control the PSO and add diversity. Extending the PSO with SOC seems promising reaching faster convergence and better solutions....

  1. Particle Swarm Optimisation with Spatial Particle Extension

    DEFF Research Database (Denmark)

    Krink, Thiemo; Vesterstrøm, Jakob Svaneborg; Riget, Jacques

    2002-01-01

    In this paper, we introduce spatial extension to particles in the PSO model in order to overcome premature convergence in iterative optimisation. The standard PSO and the new model (SEPSO) are compared w.r.t. performance on well-studied benchmark problems. We show that the SEPSO indeed managed...

  2. Topology optimisation of natural convection problems

    DEFF Research Database (Denmark)

    Alexandersen, Joe; Aage, Niels; Andreasen, Casper Schousboe

    2014-01-01

    This paper demonstrates the application of the density-based topology optimisation approach for the design of heat sinks and micropumps based on natural convection effects. The problems are modelled under the assumptions of steady-state laminar flow using the incompressible Navier-Stokes equation...

  3. Thermodynamic optimisation of a heat exchanger

    NARCIS (Netherlands)

    Cornelissen, R.L.; Hirs, G.G.

    1999-01-01

    The objective of this paper is to show that for the optimal design of an energy system, where there is a trade-off between exergy saving during operation and exergy use during construction of the energy system, exergy analysis and life cycle analysis should be combined. An exergy optimisation of a h

  4. Optimised Design of Transparent Optical Domains

    DEFF Research Database (Denmark)

    Hanik, N.; Caspar, C.; Schmidt, F.;

    2000-01-01

    Three different design concepts for transparent, dispersion compensated, optical WDM transmission links are optimised numerically and experimentally for 10 Gbit/s data rate per channel. It is shown that robust transparent domains of 1,500 km in diameter can be realised using simple design rutes....

  5. Cellular recovery from electroporation using synchronisation modulation as a rescue model for electrically injured cells.

    Science.gov (United States)

    Dando, Robin; Chen, Wei

    2008-12-01

    Electroporation of the plasma membrane resulting in a decrement in transmembrane potential is offered as a model in the study of the rescuing effects of the synchronisation modulation technique by electrically activating sodium potassium adenosine triphosphatase. Living cells were first electrically damaged by a pulsed intensive electric field, resulting in cell membrane electroporation, ion leakages and membrane potential depolarisation. Their recovery rate in natural conditions was compared with that of cells in a synchronisation modulation electric field. Fluorescence readings were taken using confocal microscopy and a potentiometric dye. Significantly more rapid recovery was observed after synchronisation modulation, with cell membranes actually polarised to levels higher than the original resting potential, a feature never seen in naturally recovering cells.

  6. In vivo imaging of cancer cells with electroporation of quantum dots and multispectral imaging

    Science.gov (United States)

    Yoo, Jung Sun; Won, Nayoun; Kim, Hong Bae; Bang, Jiwon; Kim, Sungjee; Ahn, Saeyoung; Soh, Kwang-Sup

    2010-06-01

    Our understanding of dissemination and growth of cancer cells is limited by our inability for long-term followup of this process in vivo. Fluorescence molecular imaging has the potential to track cancer cells with high contrast and sensitivity in living animals. For this purpose, intracellular delivery of near-infrared fluorescence quantum dots (QDs) by electroporation offers considerable advantages over organic fluorophores and other cell tagging methods. In this research we developed a multispectral imaging system that could eliminate two major parameters compromising in vivo fluorescence imaging performance, i.e., variations in the tissue optical properties and tissue autofluorescence. We demonstrated that electroporation of QDs and multispectral imaging allowed in vivo assessment of cancer development and progression in the xenograft mouse tumor model for more than 1 month, providing a powerful means to learn more about the biology of cancer and metastasis.

  7. Book review of: "Clinical aspects of electroporation" by Stephen T Kee, Julie Gehl, Edward W Lee

    Directory of Open Access Journals (Sweden)

    Spugnini Enrico

    2011-10-01

    Full Text Available Abstract This article is a review of the book: Clinical aspects of electroporation, by Stephen T. Kee, Julie Gehl, Edward W Lee, which is published by Springer Press. Basic information that should be helpful in deciding whether to read the book and whether to use it as a reference book is presented. This includes an introduction, a description of all the sections of the book, and a comparison with recently published books on the topic.

  8. The influence of a metal stent on the distribution of thermal energy during irreversible electroporation

    OpenAIRE

    Scheffer, Hester J.; Vogel, Jantien A.; Willemien van den Bos; Neal, Robert E; Krijn P. van Lienden; Besselink, Marc G.H.; van Gemert, Martin J. C.; van der Geld, Cees W. M.; Meijerink, Martijn R.; Klaessens, John H; Rudolf M Verdaasdonk

    2016-01-01

    Purpose Irreversible electroporation (IRE) uses short duration, high-voltage electrical pulses to induce cell death via nanoscale defects resulting from altered transmembrane potential. The technique is gaining interest for ablations in unresectable pancreatic and hepatobiliary cancer. Metal stents are often used for palliative biliary drainage in these patients, but are currently seen as an absolute contraindication for IRE due to the perceived risk of direct heating of the metal and its sur...

  9. Irreversible Electroporation of a Hepatocellular Carcinoma Lesion Adjacent to a Transjugular Intrahepatic Portosystemic Shunt Stent Graft

    Energy Technology Data Exchange (ETDEWEB)

    Niessen, Christoph; Jung, Ernst Michael; Wohlgemuth, Walter A. [Department of Radiology, University Medical Center Regensburg, Regensburg D-93053 (Germany); Trabold, Benedikt [Department of Anaesthesia, University Medical Center Regensburg, Regensburg D-93053 (Germany); Haimerl, Michael; Schreyer, Andreas; Stroszczynski, Christian; Wiggermann, Philipp [Department of Radiology, University Medical Center Regensburg, Regensburg D-93053 (Germany)

    2013-07-01

    We report in a 65-year-old man hepatocellular carcinoma adjacent to a transjugular intrahepatic portosystemic shunt stent-graft which was successfully treated with irreversible electroporation (IRE). IRE is a new non-thermal tissue ablation technique which uses electrical pulses to induce cell necrosis by irreversible membrane poration. IRE proved to be more advantageous in the ablation of perivascular tumor with little injury to the surrounding structures.

  10. Functional genomics tool: Gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA

    Directory of Open Access Journals (Sweden)

    Troiano Emily

    2010-01-01

    Full Text Available Abstract Background Ticks are blood-sucking arthropods responsible for transmitting a wide variety of disease-causing agents, and constitute important public health threats globally. Ixodes scapularis is the primary vector of the Lyme disease agent in the eastern and central U.S. RNAi is a mechanism by which gene-specific double-stranded RNA (dsRNA triggers degradation of homologous mRNA transcripts. Here, we describe an optimized protocol for effectively suppressing gene expression in the egg and nymphal stages of I. scapularis by electroporation. Results The genes encoding the putative Phospholipase A2 (PLA2, cytoplasmic Cystatin, Syntaxin-5, β-Actin and Calreticulin were targeted by delivering the dsRNA encoding the specific gene coding regions in the unfed nymphs. Silencing was measured using real time qRT-PCR. Electroporation as a mode of dsRNA delivery appears to be substantially efficient and less traumatic to the tick than dsRNA microinjection in the unfed nymphs. Using Cy3-labeled dsRNA to monitor the movement, electroporated dsRNA entered the nymphs and spread to salivary glands and other tissues. The significant disruption of β-actin and cytoplasmic Cystatin transcripts in tick eggs demonstrate the applicability of this technique. The PLA2, cytoplasmic Cystatin, Syntaxin-5, β-Actin and Calreticulin genes were also significantly silenced, suggesting that this method has the potential to introduce dsRNA in eggs and unfed nymphs. Conclusions Our study demonstrates that electroporation can be used as a simple dsRNA delivery tool in assessing the functional role of tick genes in the vector-host interactions. This technique represents a novel approach for specific gene suppression in immature stages of ticks.

  11. Inhibition of lipoxygenase activity in lentil protoplasts by monoclonal antibodies introduced into the cells via electroporation

    OpenAIRE

    J. F. G. Vliegenthart; Maccarrone, M.; Veldink, G.A.

    1992-01-01

    The isolation of lentil protoplasts and the transfer of anti-lipoxygenase monoclonal antibodies into plant protoplasts by electroporation is reported. The dependence of the efficiency of monoclonal antibody incorporation on the field strength is shown as well. The transferred immunoglobulins retained their functional and structural integrity and were able to inhibit the intracellular target enzyme, with a linear relationship between inhibition of lipoxygenase activity and amount of incorporat...

  12. Optimized delivery of fluorescently labeled proteins in live bacteria using electroporation.

    Science.gov (United States)

    Sustarsic, Marko; Plochowietz, Anne; Aigrain, Louise; Yuzenkova, Yulia; Zenkin, Nikolay; Kapanidis, Achillefs

    2014-07-01

    Studying the structure and dynamics of proteins in live cells is essential to understanding their physiological activities and mechanisms, and to validating in vitro characterization. Improvements in labeling and imaging technologies are starting to allow such in vivo studies; however, a number of technical challenges remain. Recently, we developed an electroporation-based protocol for internalization, which allows biomolecules labeled with organic fluorophores to be introduced at high efficiency into live E. coli (Crawford et al. in Biophys J 105 (11):2439-2450, 2013). Here, we address important challenges related to internalization of proteins, and optimize our method in terms of (1) electroporation buffer conditions; (2) removal of dye contaminants from stock protein samples; and (3) removal of non-internalized molecules from cell suspension after electroporation. We illustrate the usability of the optimized protocol by demonstrating high-efficiency internalization of a 10-kDa protein, the ω subunit of RNA polymerase. Provided that suggested control experiments are carried out, any fluorescently labeled protein of up to 60 kDa could be internalized using our method. Further, we probe the effect of electroporation voltage on internalization efficiency and cell viability and demonstrate that, whilst internalization increases with increased voltage, cell viability is compromised. However, due to the low number of damaged cells in our samples, the major fraction of loaded cells always corresponds to non-damaged cells. By taking care to include only viable cells into analysis, our method allows physiologically relevant studies to be performed, including in vivo measurements of protein diffusion, localization and intramolecular dynamics via single-molecule Förster resonance energy transfer.

  13. In vivo electroporation to physiologically identified deep brain regions in postnatal mammals.

    Science.gov (United States)

    Ohmura, Nami; Kawasaki, Kazuha; Satoh, Takemasa; Hata, Yoshio

    2015-01-01

    Genetic manipulation is widely used to research the central nervous system (CNS). The manipulation of molecular expression in a small number of neurons permits the detailed investigation of the role of specific molecules on the function and morphology of the neurons. Electroporation is a broadly used technique for gene transfer in the CNS. However, the targeting of gene transfer using electroporation in postnatal animals was restricted to the cortex, hippocampus, or the region facing the ventricle in previous reports. Electroporation targeting of deep brain structures, such as the thalamus, has been difficult. We introduce a novel electroporation technique that enables gene transfer to a physiologically identified deep brain region using a glass pipette. We recorded neural activity in young-adult mice to identify the location of the lateral geniculate nucleus (LGN) of the thalamus, using a glass pipette electrode containing the plasmid DNA encoding enhanced green fluorescent protein (EGFP). The location of the LGN was confirmed by monitoring visual responses, and the plasmid solution was pressure-injected into the recording site. Voltage pulses were delivered through the glass pipette electrode. Several EGFP-labeled somata and dendrites were observed in the LGN after a few weeks, and labeled axons were found in the visual cortex. The EGFP-expressing structures were observed in detail sufficient to reconstruct their morphology in three dimensions. We further confirmed the applicability of this technique in cats. This method should be useful for the transfer of various genes into cells in physiologically identified brain regions in rodents and gyrencephalic mammals.

  14. Prolonged in vivo gene silencing by electroporation-mediated plasmid delivery of small interfering RNA

    NARCIS (Netherlands)

    Eefting, D.; Grimbergen, J.M.; Vries, M.R. de; Weel, V. van; Kaijzel, E.L.; Que, I.; Moon, R.T.; Löwik, C.W.; Bockel, J.H. van; Quax, P.H.A.

    2007-01-01

    For the successful application of RNA interference in vivo, it is desired to achieve (local) delivery of small interfering RNAs (siRNAs) and long-term gene silencing. Nonviral electrodelivery is suitable to obtain local and prolonged expression of transgenes. By intramuscular electrodelivery of a pl

  15. Retroviral Vector-Mediated Gene Transfer into the Chick Optic Vesicle by In Ovo Electroporation

    Science.gov (United States)

    Sakuta, Hiraki; Suzuki, Ryoko; Noda, Masaharu

    The chick embryo offers many advantages for developmental studies over other vertebrate embryos as it allows easy access for in ovo surgical manipulations, such as tissue transplantation and the implantation of cultured cells or chemically treated beads for the local release of humoral factors. In particular, owing to its external position in the embryo, the chick eye is a popular model for studying the patterning mechanism of the central nervous system (CNS). This patterning has a crucial role in shaping functional organization because it is the basis of the specific wiring in the CNS. Genetic analysis is not easy in the chick, as compared with the mouse for which transgene introduction or gene targeting techniques have been well established. However, because methods for the expression of exogenous genes and for gene silencing in the chick embryo have been recently developed, the functional analysis of genes has become possible in combination with classical techniques of developmental biology and neurobiology.

  16. Tolerability of intramuscular and intradermal delivery by CELLECTRA® adaptive constant current electroporation device in healthy volunteers

    Science.gov (United States)

    Diehl, Malissa C; Lee, Jessica C; Daniels, Stephen E; Tebas, Pablo; Khan, Amir S; Giffear, Mary; Sardesai, Niranjan Y; Bagarazzi, Mark L

    2013-01-01

    DNA vaccines are being developed as a potentially safe and effective immunization platform. However, translation of DNA vaccines into a clinical setting has produced results that have fallen short of those generated in a preclinical setting. Various strategies are being developed to address this lack of potency, including improvements in delivery methods. Electroporation (EP) creates transient increases in cell membrane permeability, thus enhancing DNA uptake and leading to a more robust immune response. Here, we report on the safety and tolerability of delivering sterile saline via intramuscular (IM) or intradermal (ID) injection followed by in vivo electroporation using the CELLECTRA® adaptive constant current device in healthy adults from two open-label studies. Pain, as assessed by VAS, was highest immediately after EP but diminishes by about 50% within 5 min. Mean VAS scores appear to correlate with the amount of energy delivered and depth of needle insertion, especially for intramuscular EP. Mean scores did not exceed 7 out of 10 or 3 out of 10 for IM and ID EP, respectively. The majority of adverse events included mild to moderate injection site reactions that resolved within one day. No deaths or serious adverse events were reported during the course of either study. Overall, injection followed by EP with the CELLECTRA® device was well-tolerated and no significant safety concerns were identified. These studies support the further development of electroporation as a vaccine delivery method to enhance immunogenicity, particularly for diseases in which traditional vaccination approaches are ineffective. PMID:24051434

  17. Optimization of Electroporation Parameters for Immature Embryos of indica Rice(Oryza sativa)

    Institute of Scientific and Technical Information of China (English)

    REN Yu-jun; ZHAO Jie

    2008-01-01

    To obtain a suitable condition for electroporation transformation in indica rice.the 10-day-old immature embryos were selected for optimization experiments.The results showed that one pulse at 850 V/cm,950 μF capacitance,200 μL electreporation buffer with 70 mmol/L sodium glutamate,100 μg/mL plasmid,50 pg/mL carrier DNA,20 embryos per cuvette,0°C treatment and CC medium were the best parameters,which not only jmprovecl the transformation efficiency to 30.89%,but also ameliorated the embryo survival ratio to 95.92%.To further verify the practicability of this condition,the embryes from another indica rice variety and a rice type Ⅱ metallothionein-like gene(OsMT2bL)promoter::mgfp5::gusA construct were tested,and specific GUS expression on the embryos was visualized by histochemical staining.The results showed that the GUS expression on the embryos activated by the OsMT2bL promoter was mainly concentrated on the apical point of the plumule whereas the expression driven by CaMV35S promoter was distributed on nearly all areas of the electroporated tissues.These results indicated that the optimized embryo electroporation conditions could be used not only in genetic transformation of indica rice but also in assay of gene regulation on embryos.

  18. Combined Pulse Electroporation – A Novel Strategy for Highly Efficient Transfection of Human and Mouse Cells

    Science.gov (United States)

    Stroh, Thorsten; Erben, Ulrike; Kühl, Anja A.; Zeitz, Martin; Siegmund, Britta

    2010-01-01

    The type of a nucleic acid and the type of the cell to be transfected generally affect the efficiency of electroporation, the versatile method of choice for gene regulation studies or for recombinant protein expression. We here present a combined square pulse electroporation strategy to reproducibly and efficiently transfect eukaryotic cells. Cells suspended in a universal buffer system received an initial high voltage pulse that was continuously combined with a subsequent low voltage pulse with independently defined electric parameters of the effective field and the duration of each pulse. At comparable viable cell recoveries and transfection efficiencies of up to 95% of all cells, a wide variety of cells especially profited from this combined pulse strategy by high protein expression levels of individual cells after transfection. Long-term silencing of gene expression by transfected small interfering RNA was most likely due to the uptake of large nucleic acid amounts as shown by direct detection of fluorochromated small interfering RNA. The highly efficient combined pulse electroporation strategy enables for external regulation of the number of naked nucleic acid molecules taken up and can be easily adapted for cells considered difficult to transfect. PMID:20209146

  19. Combined pulse electroporation--a novel strategy for highly efficient transfection of human and mouse cells.

    Directory of Open Access Journals (Sweden)

    Thorsten Stroh

    Full Text Available The type of a nucleic acid and the type of the cell to be transfected generally affect the efficiency of electroporation, the versatile method of choice for gene regulation studies or for recombinant protein expression. We here present a combined square pulse electroporation strategy to reproducibly and efficiently transfect eukaryotic cells. Cells suspended in a universal buffer system received an initial high voltage pulse that was continuously combined with a subsequent low voltage pulse with independently defined electric parameters of the effective field and the duration of each pulse. At comparable viable cell recoveries and transfection efficiencies of up to 95% of all cells, a wide variety of cells especially profited from this combined pulse strategy by high protein expression levels of individual cells after transfection. Long-term silencing of gene expression by transfected small interfering RNA was most likely due to the uptake of large nucleic acid amounts as shown by direct detection of fluorochromated small interfering RNA. The highly efficient combined pulse electroporation strategy enables for external regulation of the number of naked nucleic acid molecules taken up and can be easily adapted for cells considered difficult to transfect.

  20. Effects of deformability and thermal motion of lipid membrane on electroporation: By molecular dynamics simulations

    KAUST Repository

    Sun, Sheng

    2011-01-01

    Effects of mechanical properties and thermal motion of POPE lipid membrane on electroporation were studied by molecular dynamics simulations. Among simulations in which specific atoms of lipids were artificially constrained at their equilibrium positions using a spring with force constant of 2.0kcal/(molÅ2) in the external electric field of 1.4kcal/(molÅe), only constraint on lateral motions of lipid tails prohibited electroporation while non-tail parts had little effects. When force constant decreased to 0.2kcal/(molÅ2) in the position constraints on lipid tails in the external electric field of 2.0kcal/(molÅe), water molecules began to enter the membrane. Position constraints of lipid tails allow water to penetrate from both sides of membrane. Thermal motion of lipids can induce initial defects in the hydrophobic core of membrane, which are favorable nucleation sites for electroporation. Simulations at different temperatures revealed that as the temperature increases, the time taken to the initial pore formation will decrease. © 2010 Elsevier Inc.

  1. High throughput transfection of cells: nano-electroporation and mobile magnetic traps

    Science.gov (United States)

    Howdyshell, M.; Gallego-Perez, D.; Vieira, G.; Malkoc, V.; Lee, L. J.; Sooryakumar, R.

    2014-03-01

    Injection of drugs or genes in vitro into cells is a critical technique for biomedical research; there are currently a number of techniques to perform such injections, but drawbacks include lack of control over dosage rates and sustained cell viability, as well as inability to inject into many cells in parallel. We have previously demonstrated a magnetically actuated nano-channel electroporation technique that multiplexes simultaneous transfection of biomolecules into cells by combining an array of remotely operated micro-magnetic traps with a nano-channel electroporation device. This device allows us to control the dosage delivered to each individual cell and reduce cell death during the experiment. The magnetic traps enable precise positioning of magnetically labeled cells and subsequent relocation of the cells for downstream processing. With this integrated approach, the number of cells transfected simultaneously has been increased nearly tenfold. In the current work, we present recent experiments with different types of cells as well as new multiplexed nano-electroporation device designs that are more high-throughput to streamline the parallel injection process, allowing the device to be implemented for a wider variety of applications.

  2. Gene Transfer from Targeted Liposomes to Specific Lymphoid Cells by Electroporation

    Science.gov (United States)

    Machy, Patrick; Lewis, Florence; McMillan, Lynette; Jonak, Zdenka L.

    1988-11-01

    Large unilamellar liposomes, coated with protein A and encapsulating the gene that confers resistance to mycophenolic acid, were used as a model system to demonstrate gene transfer into specific lymphoid cells. Protein A, which selectively recognizes mouse IgG2a antibodies, was coupled to liposomes to target them specifically to defined cell types coated with IgG2a antibody. Protein A-coated liposomes bound human B lymphoblastoid cells preincubated with a mouse IgG2a anti-HLA monoclonal antibody but failed to adhere to cells challenged with an irrelevant (anti-H-2) antibody of the same isotype or to cells incubated in the absence of antibody. Transfection of target cells bound to protein A-coated liposomes was achieved by electroporation. This step was essential since only electroporated cells survived in a selective medium containing mycophenolic acid. Transfection efficiency with electroporation and targeted liposomes was as efficient as conventional procedures that used unencapsulated plasmids free in solution but, in the latter case, cell selectivity is not possible. This technique provides a methodology for introducing defined biological macromolecules into specific cell types.

  3. Novel Parallelized Electroporation by Electrostatic Manipulation of a Water-in-Oil Droplet as a Microreactor.

    Directory of Open Access Journals (Sweden)

    Hirofumi Kurita

    Full Text Available Electroporation is the most widely used transfection method for delivery of cell-impermeable molecules into cells. We developed a novel gene transfection method, water-in-oil (W/O droplet electroporation, using dielectric oil and an aqueous droplet containing mammalian cells and transgene DNA. When a liquid droplet suspended between a pair of electrodes in dielectric oil is exposed to a DC electric field, the droplet moves between the pair of electrodes periodically and droplet deformation occurs under the intense DC electric field. During electrostatic manipulation of the droplet, the local intense electric field and instantaneous short circuit via the droplet due to droplet deformation facilitate gene transfection. This method has several advantages over conventional transfection techniques, including co-transfection of multiple transgene DNAs into even as few as 103 cells, transfection into differentiated neural cells, and the capable establishment of stable cell lines. In addition, there have been improvements in W/O droplet electroporation electrodes for disposable 96-well plates making them suitable for concurrent performance without thermal loading by a DC electric field. This technique will lead to the development of cell transfection methods for novel regenerative medicine and gene therapy.

  4. Optimization of ectopic gene expression in skeletal muscle through DNA transfer by electroporation

    Directory of Open Access Journals (Sweden)

    Latour Mickey

    2004-05-01

    Full Text Available Abstract Background Electroporation (EP is a widely used non-viral gene transfer method. We have attempted to develop an exact protocol to maximize DNA expression while minimizing tissue damage following EP of skeletal muscle in vivo. Specifically, we investigated the effects of varying injection techniques, electrode surface geometry, and plasmid mediums. Results We found that as the amount of damage increased in skeletal muscle in response to EP, the level of β-galactosidase (β-gal expression drastically decreased and that there was no evidence of β-gal expression in damaged fibers. Two specific types of electrodes yielded the greatest amount of expression. We also discovered that DNA uptake in skeletal muscle following intra-arterial injection of DNA was significantly enhanced by EP. Finally, we found that DMSO and LipoFECTAMINE™, common enhancers of DNA electroporation in vitro, had no positive effect on DNA electroporation in vivo. Conclusions When injecting DNA intramuscularly, a flat plate electrode without any plasmid enhancers is the best method to achieve high levels of gene expression.

  5. Intramuscular electroporation of a P1A-encoding plasmid vaccine delays P815 mastocytoma growth.

    Science.gov (United States)

    Vandermeulen, Gaëlle; Uyttenhove, Catherine; De Plaen, Etienne; Van den Eynde, Benoît J; Préat, Véronique

    2014-12-01

    This study aimed to construct DNA vaccines encoding the mouse P1A tumor antigen and to generate a protective immune response against the P815 mastocytoma, as a model for vaccines against human MAGE-type tumor antigens. DNA vaccines were constructed and delivered to mice by intramuscular electroporation before tumor challenge. Immunization with a plasmid coding for the full-length P1A significantly delayed tumor growth and mice survived at least 10 days longer than untreated controls. 10% of the mice completely rejected the P815 tumors while 50% of them showed a regression phase followed by tumor regrowth. Mice immunized by electroporation of a P1A(35-43) minigene-encoding plasmid failed to reject tumor and even delay tumor growth. The P1A(35-43)-encoding plasmid was modified and helper epitope sequences were inserted. However, these modified plasmids were not able to improve the response against P815 mastocytoma. Consistent with these results, a 12-fold higher CTL activity was observed when the plasmid coding for full-length P1A was delivered as compared to the plasmid encoding the P1A(35-43) epitope. Our results demonstrated that electroporation is an efficient method to deliver DNA vaccines against P815 and suggested the superiority of full-length as compared to minigene constructs for DNA vaccines.

  6. Modelling the Bioelectronic Interface in Engineered Tethered Membranes: From Biosensing to Electroporation.

    Science.gov (United States)

    Hoiles, William; Krishnamurthy, Vikram; Cornell, Bruce

    2015-06-01

    This paper studies the construction and predictive models of three novel measurement platforms: (i) a Pore Formation Measurement Platform (PFMP) for detecting the presence of pore forming proteins and peptides, (ii) the Ion Channel Switch (ICS) biosensor for detecting the presence of analyte molecules in a fluid chamber, and (iii) an Electroporation Measurement Platform (EMP) that provides reliable measurements of the electroporation phenomenon. Common to all three measurement platforms is that they are comprised of an engineered tethered membrane that is formed via a rapid solvent exchange technique allowing the platform to have a lifetime of several months. The membrane is tethered to a gold electrode bioelectronic interface that includes an ionic reservoir separating the membrane and gold surface, allowing the membrane to mimic the physiological response of natural cell membranes. The electrical response of the PFMP, ICS, and EMP are predicted using continuum theories for electrodiffusive flow coupled with boundary conditions for modelling chemical reactions and electrical double layers present at the bioelectronic interface. Experimental measurements are used to validate the predictive accuracy of the dynamic models. These include using the PFMP for measuring the pore formation dynamics of the antimicrobial peptide PGLa and the protein toxin Staphylococcal α-Hemolysin; the ICS biosensor for measuring nano-molar concentrations of streptavidin, ferritin, thyroid stimulating hormone (TSH), and human chorionic gonadotropin (pregnancy hormone hCG); and the EMP for measuring electroporation of membranes with different tethering densities, and membrane compositions.

  7. Interpulse multifrequency electrical impedance measurements during electroporation of adherent differentiated myotubes.

    Science.gov (United States)

    García-Sánchez, Tomás; Azan, Antoine; Leray, Isabelle; Rosell-Ferrer, Javier; Bragós, Ramon; Mir, Lluis M

    2015-10-01

    In this study, electrical impedance spectroscopy measurements are performed during electroporation of monolayers of differentiated myotubes. The time resolution of the system (1 spectrum/ms) enable 860 full spectra (21 frequencies from 5 kHz to 1.3 MHz) to be acquired during the time gap between consecutive pulses (interpulse) of a classical electroporation treatment (8 pulses, 100 μs, 1 Hz). Additionally, the characteristics of the custom microelectrode assembly used allow the experiments to be performed directly in situ in standard 24 multi-well plates. The impedance response dynamics are studied for three different electric field intensities (400, 800 and 1200 V/cm). The multifrequency information, analysed with the Cole model, reveals a short-term impedance recovery after each pulse in accordance with the fast resealing of the cell membrane, and a long-term impedance decay over the complete treatment in accordance with an accumulated effect pulse after pulse. The analysis shows differences between the lowest electric field condition and the other two, suggesting that different mechanisms that may be related with the reversibility of the process are activated. As a result of the multifrequency information, the system is able to measure simultaneously the conductivity variations due to ion diffusion during electroporation. Finally, in order to reinforce the physical interpretation of the results, a complementary electrical equivalent model is used.

  8. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control.

    Science.gov (United States)

    Chang, Lingqian; Bertani, Paul; Gallego-Perez, Daniel; Yang, Zhaogang; Chen, Feng; Chiang, Chiling; Malkoc, Veysi; Kuang, Tairong; Gao, Keliang; Lee, L James; Lu, Wu

    2016-01-01

    Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a "3D nano-channel electroporation (NEP) chip" on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40,000 cells per cm(2) on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells.

  9. A Nonlinear Size-Dependent Equivalent Circuit Model for Single-Cell Electroporation on Microfluidic Chips.

    Science.gov (United States)

    Shagoshtasbi, Hooman; Deng, Peigang; Lee, Yi-Kuen

    2015-08-01

    Electroporation (EP) is a process of applying a pulsed intense electric field on the cell membrane to temporarily induce nanoscale electropores on the plasma membrane of biological cells. A nonlinear size-dependent equivalent circuit model of a single-cell electroporation system is proposed to investigate dynamic electromechanical behavior of cells on microfluidic chips during EP. This model consists of size-dependent electromechanical components of a cell, electrical components of poration media, and a microfluidic chip. A single-cell microfluidic EP chip with 3D microelectrode arrays along a microchannel is designed and fabricated to experimentally analyze the permeabilization of a cell. Predicted electrical current responses of the model are in good agreement (average error of 6%) with that of single-cell EP. The proposed model can successfully predict the time responses of transmembrane voltage, pore diameter, and pore density at four different stages of permeabilization. These stages are categorized based on electromechanical changes of the lipid membrane. The current-voltage characteristic curve of the cell membrane during EP is also investigated at different EP stages in detail. The model can precisely predict the electric breakdown of different cell lines at a specific critical cell membrane voltage of the target cell lines.

  10. Improved electroporation procedure for genetic transformation of Dekkera/Brettanomyces bruxellensis.

    Science.gov (United States)

    Miklenić, Marina; Žunar, Bojan; Štafa, Anamarija; Svetec, Ivan-Krešimir

    2015-12-01

    Yeast Dekkera/Brettanomyces bruxellensis is one of the most common contaminants in wine industry, but also one of the most promising candidates for large-scale bioethanol production. Brettanomyces bruxellensis not only produces and tolerates high ethanol concentrations, but can also ferment cellobiose and adapt to lignocellulose hydrolasate. Furthermore, genome sequences of several B. bruxellensis strains are available, and efforts have been made to develop tools for genetic transformation of this yeast. Previously, we reported a successful transformation using lithium acetate/PEG method and electroporation, however, with very low transformation efficiency (10-20 transformants μg(-1)). Here we describe an optimization of electroporation procedure which resulted in a significant increase of transformation efficiency (2.8 × 10(3) transformants μg(-1)). Several key transformation parameters were optimized including cell growth phase, density of cells in the transformation sample and electroporation settings. We determined that treating the cells with both lithium acetate (100 mM) and dithiothreitol (35 mM) synergistically improves transformation efficiency. Using the described procedure around 500 transformants can be obtained per transformation sample with 180 ng of non-homologous linear transforming fragment. Additionally, several transformants were obtained with less than 1 ng of DNA demonstrating that this procedure is adequate even when very limited amount of DNA is available. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. The effect of small intestine heterogeneity on irreversible electroporation treatment planning.

    Science.gov (United States)

    Phillips, Mary

    2014-09-01

    Nonthermal irreversible electroporation (NTIRE) is an ablation modality that utilizes microsecond electric fields to produce nanoscale defects in the cell membrane. This results in selective cell death while preserving all other molecules, including the extracellular matrix. Here, finite element analysis and experimental results are utilized to examine the effect of NTIRE on the small intestine due to concern over collateral damage to this organ during NTIRE treatment of abdominal cancers. During previous studies, the electrical treatment parameters were chosen based on a simplified homogeneous tissue model. The small intestine, however, has very distinct layers, and a more realistic model is needed to further develop this technology for precise clinical applications. This study uses a two-dimensional finite element solution of the Laplace and heat conduction equations to investigate how small intestine heterogeneities affect the electric field and temperature distribution. Experimental results obtained by applying NTIRE to the rat small intestine in vivo support the heterogeneous effect of NTIRE on the tissue. The numerical modeling indicates that the electroporation parameters chosen for this study avoid thermal damage to the tissue. This is supported by histology obtained from the in vivo study, which showed preservation of extracellular structures. The finite element model also indicates that the heterogeneous structure of the small intestine has a significant effect on the electric field and volume of cell ablation during electroporation and could have a large impact on the extent of treatment. The heterogeneous nature of the tissue should be accounted for in clinical treatment planning.

  12. Skin Transfection Patterns and Expression Kinetics of Electroporation-Enhanced Plasmid Delivery Using the CELLECTRA-3P, a Portable Next-Generation Dermal Electroporation Device.

    Science.gov (United States)

    Amante, Dinah H; Smith, Trevor R F; Mendoza, Janess M; Schultheis, Katherine; McCoy, Jay R; Khan, Amir S; Sardesai, Niranjan Y; Broderick, Kate E

    2015-08-01

    The CELLECTRA-3P dermal electroporation device (Inovio Pharmaceuticals, Plymouth Meeting, PA) has been evaluated in the clinic and shown to enhance the delivery of an influenza DNA vaccine. To understand the mechanism by which this device aids in enhancing the host immune response to DNA vaccines we investigated the expression kinetics and localization of a reporter plasmid (pGFP) delivered via the CELLECTRA-3P. Histological analysis revealed green fluorescent protein (GFP) expression as early as 1 hr posttreatment in the epidermal and dermal layers, and as early as 2 hr posttreatment in the subdermal layers. Immunofluorescence techniques identified keratinocytes, fibrocytes, dendritic-like cells, adipocytes, and myocytes as the principal cell populations transfected. We proceeded to demonstrate elicitation of robust host immune responses after plasmid DNA (pDNA) vaccination. In guinea pigs equivalent humoral (antibody binding titers) immune responses were observed between protocols using either CELLECTRA-3P or intramuscular electroporation to deliver the DNA vaccine. In nonhuman primates, robust interferon-γ enzyme-linked immunospot and protective levels of hemagglutination inhibition titers after pDNA vaccination were observed in groups treated with the CELLECTRA-3P. In conclusion, these findings may assist in the future to design efficient, tolerable DNA vaccination strategies for the clinic.

  13. A numerical investigation of the electric and thermal cell kill distributions in electroporation-based therapies in tissue.

    Directory of Open Access Journals (Sweden)

    Paulo A Garcia

    Full Text Available Electroporation-based therapies are powerful biotechnological tools for enhancing the delivery of exogeneous agents or killing tissue with pulsed electric fields (PEFs. Electrochemotherapy (ECT and gene therapy based on gene electrotransfer (EGT both use reversible electroporation to deliver chemotherapeutics or plasmid DNA into cells, respectively. In both ECT and EGT, the goal is to permeabilize the cell membrane while maintaining high cell viability in order to facilitate drug or gene transport into the cell cytoplasm and induce a therapeutic response. Irreversible electroporation (IRE results in cell kill due to exposure to PEFs without drugs and is under clinical evaluation for treating otherwise unresectable tumors. These PEF therapies rely mainly on the electric field distributions and do not require changes in tissue temperature for their effectiveness. However, in immediate vicinity of the electrodes the treatment may results in cell kill due to thermal damage because of the inhomogeneous electric field distribution and high current density during the electroporation-based therapies. Therefore, the main objective of this numerical study is to evaluate the influence of pulse number and electrical conductivity in the predicted cell kill zone due to irreversible electroporation and thermal damage. Specifically, we simulated a typical IRE protocol that employs ninety 100-µs PEFs. Our results confirm that it is possible to achieve predominant cell kill due to electroporation if the PEF parameters are chosen carefully. However, if either the pulse number and/or the tissue conductivity are too high, there is also potential to achieve cell kill due to thermal damage in the immediate vicinity of the electrodes. Therefore, it is critical for physicians to be mindful of placement of electrodes with respect to critical tissue structures and treatment parameters in order to maintain the non-thermal benefits of electroporation and prevent

  14. A numerical investigation of the electric and thermal cell kill distributions in electroporation-based therapies in tissue.

    Science.gov (United States)

    Garcia, Paulo A; Davalos, Rafael V; Miklavcic, Damijan

    2014-01-01

    Electroporation-based therapies are powerful biotechnological tools for enhancing the delivery of exogeneous agents or killing tissue with pulsed electric fields (PEFs). Electrochemotherapy (ECT) and gene therapy based on gene electrotransfer (EGT) both use reversible electroporation to deliver chemotherapeutics or plasmid DNA into cells, respectively. In both ECT and EGT, the goal is to permeabilize the cell membrane while maintaining high cell viability in order to facilitate drug or gene transport into the cell cytoplasm and induce a therapeutic response. Irreversible electroporation (IRE) results in cell kill due to exposure to PEFs without drugs and is under clinical evaluation for treating otherwise unresectable tumors. These PEF therapies rely mainly on the electric field distributions and do not require changes in tissue temperature for their effectiveness. However, in immediate vicinity of the electrodes the treatment may results in cell kill due to thermal damage because of the inhomogeneous electric field distribution and high current density during the electroporation-based therapies. Therefore, the main objective of this numerical study is to evaluate the influence of pulse number and electrical conductivity in the predicted cell kill zone due to irreversible electroporation and thermal damage. Specifically, we simulated a typical IRE protocol that employs ninety 100-µs PEFs. Our results confirm that it is possible to achieve predominant cell kill due to electroporation if the PEF parameters are chosen carefully. However, if either the pulse number and/or the tissue conductivity are too high, there is also potential to achieve cell kill due to thermal damage in the immediate vicinity of the electrodes. Therefore, it is critical for physicians to be mindful of placement of electrodes with respect to critical tissue structures and treatment parameters in order to maintain the non-thermal benefits of electroporation and prevent unnecessary damage to

  15. Assessment and optimization of electroporation-assisted tumoral nanoparticle uptake in a nude mouse model of pancreatic ductal adenocarcinoma.

    Science.gov (United States)

    West, Derek Lamont; White, Sarah B; Zhang, Zhouli; Larson, Andrew C; Omary, Reed A

    2014-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is a particularly lethal form of cancer. In 2012, the incidence of PDAC was 43,920. Five-year survival for patients with PDAC is around 6%, regardless of staging, making PDAC one of the deadliest forms of cancer. One reason for this dismal prognosis is chemoresistance to the current first-line therapy, gemcitabine. There are multiple factors that contribute to the chemoresistance observed in pancreatic cancer. Among them, desmoplasia has been increasingly seen as a significant contributor to chemoresistance. To overcome desmoplastic chemoresistance, several novel methods of treatment have been developed. Electroporation is one such novel treatment. High electrical fields are applied to cells to create pores that increase cell permeability. It has been previously demonstrated that electroporation enhances the therapeutic efficacy of anticancer drugs in pancreatic tumor models. Nanoparticle-based drug delivery systems constitute a second novel method to overcome desmoplastic chemoresistance. Due to their intrinsic design advantages, nanoparticles have been shown to increase the effectiveness of chemotherapeutic agents, while further reducing or even eliminating side effects. To date, there have been no studies evaluating the cumulative effect of combining both nanoparticle and electroporation strategies to overcome chemoresistance in PDAC. Our preliminary studies assessed the in vitro and in vivo uptake of doxorubicin-loaded iron oxide nanoparticles as a function of electroporation voltage and timing of administration in pancreatic adenocarcinoma cells. Our studies demonstrated that addition of electroporation to administration of nanoparticles significantly increased the amount of intracellular iron oxide nanoparticle uptake by a PANC-1 cell line in an athymic nude mouse model of PDAC. Further, electroporation-assisted nanoparticle uptake could be significantly altered by changing the timing of application of electroporation.

  16. Optimization of The Electroporation Conditions for Transfection of Human Factor IX into The Goat Fetal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Amir Amiri Yekta

    2013-01-01

    Full Text Available Objective: Electroporation is the most common method used for the transfection of DNA. Although this method has been attributed for various cell using different buffer compositions, the effects of DNA concentration on the transfection efficiency has not yet been studied. Here the correlation between the efficiency of electroporation reaction and increments of DNA concentration has been investigated. Following this investigation, a study was set out to produce a transgenic goat which is capable of secreting recombinant human coagulation factor IX in its milk.Materials and Methods: In this experimental study, a linearized recombinant vector (pBC1 entailing human coagulation factor IX (5.7 kb cDNA was introduced into goat fetal fibroblast cells (three sub passages via electroporation in four separate experiments (while other variables were kept constant, beginning with 10 μg DNA per pulse in the first experiment and increments of 10 μg/pulse for the next three reactions. Thereafter, polymerase chain reaction (PCR-positive cell culture plates were diluted by several factors for further analysis of the transfected wells. Subsequently, positive cells were isolated for fluorescence in situ hybridization. Logistic regression model was used for data analyzing. Significance was defined as p< 0.05.Results: The results showed no significant difference among three first concentrations except for group 1 (10 μg/pulse and group 3 (30 μg/pulse, but there was a significant difference between these groups and the fourth group (p<0.05, as this group (40 μg/pulse statistically showed the highest rate of transfection. As the fluorescence in situ hybridization (FISH results indicated the transgene was integrated in a single position in PCR positive cells.Conclusion: Increasing amount of using the vector 40μg/pulse efficiently increased the number of transfected cells. Besides of voltage and buffer strength which had been previously shown to play a critical role

  17. Education on electrical phenomena involved in electroporation-based therapies and treatments: a blended learning approach.

    Science.gov (United States)

    Čorović, Selma; Mahnič-Kalamiza, Samo; Miklavčič, Damijan

    2016-04-07

    Electroporation-based applications require multidisciplinary expertise and collaboration of experts with different professional backgrounds in engineering and science. Beginning in 2003, an international scientific workshop and postgraduate course electroporation based technologies and treatments (EBTT) has been organized at the University of Ljubljana to facilitate transfer of knowledge from leading experts to researches, students and newcomers in the field of electroporation. In this paper we present one of the integral parts of EBTT: an e-learning practical work we developed to complement delivery of knowledge via lectures and laboratory work, thus providing a blended learning approach on electrical phenomena involved in electroporation-based therapies and treatments. The learning effect was assessed via a pre- and post e-learning examination test composed of 10 multiple choice questions (i.e. items). The e-learning practical work session and both of the e-learning examination tests were carried out after the live EBTT lectures and other laboratory work. Statistical analysis was performed to compare and evaluate the learning effect measured in two groups of students: (1) electrical engineers and (2) natural scientists (i.e. medical doctors, biologists and chemists) undergoing the e-learning practical work in 2011-2014 academic years. Item analysis was performed to assess the difficulty of each item of the examination test. The results of our study show that the total score on the post examination test significantly improved and the item difficulty in both experimental groups decreased. The natural scientists reached the same level of knowledge (no statistical difference in total post-examination test score) on the post-course test take, as do electrical engineers, although the engineers started with statistically higher total pre-test examination score, as expected. The main objective of this study was to investigate whether the educational content the e

  18. Mitigation of impedance changes due to electroporation therapy using bursts of high-frequency bipolar pulses.

    Science.gov (United States)

    Bhonsle, Suyashree P; Arena, Christopher B; Sweeney, Daniel C; Davalos, Rafael V

    2015-01-01

    For electroporation-based therapies, accurate modeling of the electric field distribution within the target tissue is important for predicting the treatment volume. In response to conventional, unipolar pulses, the electrical impedance of a tissue varies as a function of the local electric field, leading to a redistribution of the field. These dynamic impedance changes, which depend on the tissue type and the applied electric field, need to be quantified a priori, making mathematical modeling complicated. Here, it is shown that the impedance changes during high-frequency, bipolar electroporation therapy are reduced, and the electric field distribution can be approximated using the analytical solution to Laplace's equation that is valid for a homogeneous medium of constant conductivity. Two methods were used to examine the agreement between the analytical solution to Laplace's equation and the electric fields generated by 100 µs unipolar pulses and bursts of 1 µs bipolar pulses. First, pulses were applied to potato tuber tissue while an infrared camera was used to monitor the temperature distribution in real-time as a corollary to the electric field distribution. The analytical solution was overlaid on the thermal images for a qualitative assessment of the electric fields. Second, potato ablations were performed and the lesion size was measured along the x- and y-axes. These values were compared to the analytical solution to quantify its ability to predict treatment outcomes. To analyze the dynamic impedance changes due to electroporation at different frequencies, electrical impedance measurements (1 Hz to 1 MHz) were made before and after the treatment of potato tissue. For high-frequency bipolar burst treatment, the thermal images closely mirrored the constant electric field contours. The potato tissue lesions differed from the analytical solution by 39.7 ± 1.3 % (x-axis) and 6.87 ± 6.26 % (y-axis) for conventional unipolar pulses, and 15.46 ± 1.37 % (x

  19. 3D Nanochannel Array Platform for High-throughput Cell Manipulation and Nano-electroporation

    Science.gov (United States)

    Chang, Lingqian

    Electroporation is one of the most common non-viral methods for gene delivery. Recent progress in gene therapy has offered special opportunities to electroporation for in vitro and in vivo applications. However, conventional bulk electroporation (BEP) inevitably causes serious cell damage and stochastic transfection between cells. Microfluidic electroporation (MEP) has been claimed to provide benign single cell transfection for the last decade. Nevertheless, the intracellular transport in both MEP and BEP systems is highly diffusion-dominant, which prevents precise dose control and high uniformity. In this Ph.D. research, we developed a 3D nanochannel-electroporation (3D NEP) platform for mass cell transfection. A silicon-based nanochannel array (3D NEP) chip was designed and fabricated for cell manipulation and electroporation. The chip, designed as Z-directional microchannel - nanochannel array, was fabricated by clean room techniques including projection photolithography and deep reactive-ion etching (DRIE). The fabricated 3D NEP chip is capable of handling 40,000 cells per 1 cm2, up to 1 million per wafer (100 mm diameter). High-throughput cell manipulation technologies were investigated for precise alignment of individual cells to the nanochannel array, a key step for NEP to achieve dose control. We developed three techniques for cell trapping in this work. (1) Magnetic tweezers (MTs) were integrated on the chip to remotely control cells under a programmed magnetic field. (2) A positive dielectrophoresis (pDEP) power system was built as an alternative to trap cells onto the nanochannel array using DEP force. (3) A novel yet simple 'dipping-trap' method was used to rapidly trap cells onto a nanochannel array, aligned by a micro-cap array pattern on the 3D NEP chip, which eventually offered 70 - 90 % trapping efficiency and 90 % specificity. 3D NEP platforms were assembled for cell transfection based on the Si-based nanochannel array chip and cell manipulation

  20. Public transport optimisation emphasising passengers’ travel behaviour

    DEFF Research Database (Denmark)

    Jensen, Jens Parbo

    to enhance the operations of public transport while explicitly emphasising passengers’ travel behaviour and preferences. Similar to economic theory, interactions between supply and demand are omnipresent in the context of public transport operations. In public transport, the demand is represented...... the published performance measures and what passengers actually experience, a large academic contribution of the current PhD study is the explicit consideration of passengers’ travel behaviour in optimisation studies and in the performance assessment. Besides the explicit passenger focus in transit planning...... at as the motivator for delay-robust railway timetables. Interestingly, passenger oriented optimisation studies considering robustness in railway planning typically limit their emphasis on passengers to the consideration of transfer maintenance. Clearly, passengers’ travel behaviour is more complex and multifaceted...

  1. Topology optimised planar photonic crystal building blocks

    DEFF Research Database (Denmark)

    Frandsen, Lars Hagedorn; Hede, K. K.; Borel, Peter Ingo

    A photonic crystal waveguide (PhCW) 1x4 splitter has been constructed from PhCW 60° bends1 and Y-splitters2 that have been designed individually by utilising topology optimisation3. The splitter has been fabricated in a silicon-on-insulator material (Fig. 1) and exhibits a broadband splitting...... for the TE-polarisation with an average excess loss of 1.55±0.54 dB for a 110 nm bandwidth. The 1x4 splitter demonstrates that individual topology-optimised parts can be used as building blocks to realise high-performance nanophotonic circuits. 1L. H. Frandsen et al., Opt. Express 12, 5916-5921 (2004) 2P. I...

  2. Self-optimising control of sewer systems

    DEFF Research Database (Denmark)

    Mauricio Iglesias, Miguel; Montero-Castro, I.; Mollerup, Ane Loft

    2013-01-01

    Self-optimising control is a useful concept to select optimal controlled variables from a set of candidate measurements in a systematic manner. In this study, use self-optimizing control tools and apply them to the specific features of sewer systems, e.g. the continuously transient dynamics......, the availability of a large number of measurements, the stochastic and unforeseeable character of the disturbances (rainfall). Using a subcatchment area in the Copenhagen sewer system as a case study we demonstrate, step by step, the formulation of the self-optimising control problem. The final result...... is an improved control structure aimed at optimizing the losses for a given control objective, here the minimization of the combined sewer overflows despite rainfall variations....

  3. Improved Squeaky Wheel Optimisation for Driver Scheduling

    CERN Document Server

    Aickelin, Uwe; Li, Jingpeng

    2008-01-01

    This paper presents a technique called Improved Squeaky Wheel Optimisation for driver scheduling problems. It improves the original Squeaky Wheel Optimisations effectiveness and execution speed by incorporating two additional steps of Selection and Mutation which implement evolution within a single solution. In the ISWO, a cycle of Analysis-Selection-Mutation-Prioritization-Construction continues until stopping conditions are reached. The Analysis step first computes the fitness of a current solution to identify troublesome components. The Selection step then discards these troublesome components probabilistically by using the fitness measure, and the Mutation step follows to further discard a small number of components at random. After the above steps, an input solution becomes partial and thus the resulting partial solution needs to be repaired. The repair is carried out by using the Prioritization step to first produce priorities that determine an order by which the following Construction step then schedul...

  4. Buckling optimisation of sandwich cylindrical panels

    Science.gov (United States)

    Abouhamzeh, M.; Sadighi, M.

    2016-06-01

    In this paper, the buckling load optimisation is performed on sandwich cylindrical panels. A finite element program is developed in MATLAB to solve the governing differential equations of the global buckling of the structure. In order to find the optimal solution, the genetic algorithm Toolbox in MATLAB is implemented. Verifications are made for both the buckling finite element code and also the results from the genetic algorithm by comparisons to the results available in literature. Sandwich cylindrical panels are optimised for the buckling strength with isotropic or orthotropic cores with different boundary conditions. Results are presented in terms of stacking sequence of fibers in the face sheets and core to face sheet thickness ratio.

  5. Applying the Theory of Optimising Professional Life

    Directory of Open Access Journals (Sweden)

    Lesley Margaret Piko

    2014-12-01

    Full Text Available Glaser (2014 wrote that “the application of grounded theory (GT is a relatively neglected topic” (p. 1 in the literature. Applying GT to purposely intervene and improve a situation is an important adjunct to our knowledge and understanding of GT. A recent workshop of family doctors and general practitioners provides a useful example. The theory of optimising professional life explains that doctors are concerned about sustainment in their career and, to resolve this concern, they implement solutions to optimise their personal situation. Sustainment is a new, overarching concept of three needs: the need for self-care to sustain well-being, the need for work interest to sustain motivation, and the need for income to sustain lifestyle. The objective of the workshop was to empower doctors to reinvent their careers using this theory. Working individually and in small groups, participants were able to analyse a problem and to identify potential solutions.

  6. Fermionic orbital optimisation in tensor network states

    CERN Document Server

    Krumnow, C; Eisert, J

    2015-01-01

    Tensor network states and specifically matrix-product states have proven to be a powerful tool for simulating ground states of strongly correlated spin models. Recently, they have also been applied to interacting fermionic problems, specifically in the context of quantum chemistry. A new freedom arising in such non-local fermionic systems is the choice of orbitals, it being far from clear what choice of fermionic orbitals to make. In this work, we propose a way to overcome this challenge. We suggest a method intertwining the optimisation over matrix product states with suitable fermionic Gaussian mode transformations, hence bringing the advantages of both approaches together. The described algorithm generalises basis changes in the spirit of the Hartree-Fock methods to matrix-product states, and provides a black box tool for basis optimisations in tensor network methods.

  7. Adaptive Java Optimisation using machine learning techniques

    OpenAIRE

    Long, Shun

    2004-01-01

    There is a continuing demand for higher performance, particularly in the area of scientific and engineering computation. In order to achieve high performance in the context of frequent hardware upgrading, software must be adaptable for portable performance. What is required is an optimising compiler that evolves and adapts itself to environmental change without sacrificing performance. Java has emerged as a dominant programming language widely used in a variety of application areas. Howeve...

  8. ATLAS software configuration and build tool optimisation

    Science.gov (United States)

    Rybkin, Grigory; Atlas Collaboration

    2014-06-01

    ATLAS software code base is over 6 million lines organised in about 2000 packages. It makes use of some 100 external software packages, is developed by more than 400 developers and used by more than 2500 physicists from over 200 universities and laboratories in 6 continents. To meet the challenge of configuration and building of this software, the Configuration Management Tool (CMT) is used. CMT expects each package to describe its build targets, build and environment setup parameters, dependencies on other packages in a text file called requirements, and each project (group of packages) to describe its policies and dependencies on other projects in a text project file. Based on the effective set of configuration parameters read from the requirements files of dependent packages and project files, CMT commands build the packages, generate the environment for their use, or query the packages. The main focus was on build time performance that was optimised within several approaches: reduction of the number of reads of requirements files that are now read once per package by a CMT build command that generates cached requirements files for subsequent CMT build commands; introduction of more fine-grained build parallelism at package task level, i.e., dependent applications and libraries are compiled in parallel; code optimisation of CMT commands used for build; introduction of package level build parallelism, i. e., parallelise the build of independent packages. By default, CMT launches NUMBER-OF-PROCESSORS build commands in parallel. The other focus was on CMT commands optimisation in general that made them approximately 2 times faster. CMT can generate a cached requirements file for the environment setup command, which is especially useful for deployment on distributed file systems like AFS or CERN VMFS. The use of parallelism, caching and code optimisation significantly-by several times-reduced software build time, environment setup time, increased the efficiency of

  9. Optimised polarisation measurements on Bragg peaks

    Energy Technology Data Exchange (ETDEWEB)

    Lelievre-Berna, E. [Institut Laue Langevin, 6 rue Jules Horowitz, 38042 Grenoble Cedex 9 (France)]. E-mail: lelievre@ill.fr; Brown, P.J. [Institut Laue Langevin, 6 rue Jules Horowitz, 38042 Grenoble Cedex 9 (France); Tasset, F. [Institut Laue Langevin, 6 rue Jules Horowitz, 38042 Grenoble Cedex 9 (France)

    2007-07-15

    Experimentally the asymmetry A (or the flipping ratio R) is deduced from the two count rates observed for |+> and |-> neutron spin states. Since the count rates for the two spin states may be quite different and both require to be corrected for background, the optimum strategy for the measurement is important. We present here the theory for optimisation of the accuracy of measurement of A (or R) within the constraint of a fixed total measuring time.

  10. Electroporated Antigen-Encoding mRNA Is Not a Danger Signal to Human Mature Monocyte-Derived Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Stefanie Hoyer

    2015-01-01

    Full Text Available For therapeutic cancer vaccination, the adoptive transfer of mRNA-electroporated dendritic cells (DCs is frequently performed, usually with monocyte-derived, cytokine-matured DCs (moDCs. However, DCs are rich in danger-sensing receptors which could recognize the exogenously delivered mRNA and induce DC activation, hence influencing the DCs’ immunogenicity. Therefore, we examined whether electroporation of mRNA with a proper cap and a poly-A tail of at least 64 adenosines had any influence on cocktail-matured moDCs. We used 16 different RNAs, encoding tumor antigens (MelanA, NRAS, BRAF, GNAQ, GNA11, and WT1, and variants thereof. None of those RNAs induced changes in the expression of CD25, CD40, CD83, CD86, and CD70 or the secretion of the cytokines IL-8, IL-6, and TNFα of more than 1.5-fold compared to the control condition, while an mRNA encoding an NF-κB-activation protein as positive control induced massive secretion of the cytokines. To determine whether mRNA electroporation had any effect on the whole transcriptome of the DCs, we performed microarray analyses of DCs of 6 different donors. None of 60,000 probes was significantly different between mock-electroporated DCs and MelanA-transfected DCs. Hence, we conclude that no transcriptional programs were induced within cocktail-matured DCs by electroporation of single tumor-antigen-encoding mRNAs.

  11. Whole tumor antigen vaccination using dendritic cells: Comparison of RNA electroporation and pulsing with UV-irradiated tumor cells

    Directory of Open Access Journals (Sweden)

    Benencia Fabian

    2008-04-01

    Full Text Available Abstract Because of the lack of full characterization of tumor associated antigens for solid tumors, whole antigen use is a convenient approach to tumor vaccination. Tumor RNA and apoptotic tumor cells have been used as a source of whole tumor antigen to prepare dendritic cell (DC based tumor vaccines, but their efficacy has not been directly compared. Here we compare directly RNA electroporation and pulsing of DCs with whole tumor cells killed by ultraviolet (UV B radiation using a convenient tumor model expressing human papilloma virus (HPV E6 and E7 oncogenes. Although both approaches led to DCs presenting tumor antigen, electroporation with tumor cell total RNA induced a significantly higher frequency of tumor-reactive IFN-gamma secreting T cells, and E7-specific CD8+ lymphocytes compared to pulsing with UV-irradiated tumor cells. DCs electroporated with tumor cell RNA induced a larger tumor infiltration by T cells and produced a significantly stronger delay in tumor growth compared to DCs pulsed with UV-irradiated tumor cells. We conclude that electroporation with whole tumor cell RNA and pulsing with UV-irradiated tumor cells are both effective in eliciting antitumor immune response, but RNA electroporation results in more potent tumor vaccination under the examined experimental conditions.

  12. Ex Vivo and In Silico Feasibility Study of Monitoring Electric Field Distribution in Tissue during Electroporation Based Treatments

    Science.gov (United States)

    Kranjc, Matej; Bajd, Franci; Sersa, Igor; Woo, Eung Je; Miklavcic, Damijan

    2012-01-01

    Magnetic resonance electrical impedance tomography (MREIT) was recently proposed for determining electric field distribution during electroporation in which cell membrane permeability is temporary increased by application of an external high electric field. The method was already successfully applied for reconstruction of electric field distribution in agar phantoms. Before the next step towards in vivo experiments is taken, monitoring of electric field distribution during electroporation of ex vivo tissue ex vivo and feasibility for its use in electroporation based treatments needed to be evaluated. Sequences of high voltage pulses were applied to chicken liver tissue in order to expose it to electric field which was measured by means of MREIT. MREIT was also evaluated for its use in electroporation based treatments by calculating electric field distribution for two regions, the tumor and the tumor-liver region, in a numerical model based on data obtained from clinical study on electrochemotherapy treatment of deep-seated tumors. Electric field distribution inside tissue was successfully measured ex vivo using MREIT and significant changes of tissue electrical conductivity were observed in the region of the highest electric field. A good agreement was obtained between the electric field distribution obtained by MREIT and the actual electric field distribution in evaluated regions of a numerical model, suggesting that implementation of MREIT could thus enable efficient detection of areas with insufficient electric field coverage during electroporation based treatments, thus assuring the effectiveness of the treatment. PMID:23029212

  13. Ex vivo and in silico feasibility study of monitoring electric field distribution in tissue during electroporation based treatments.

    Directory of Open Access Journals (Sweden)

    Matej Kranjc

    Full Text Available Magnetic resonance electrical impedance tomography (MREIT was recently proposed for determining electric field distribution during electroporation in which cell membrane permeability is temporary increased by application of an external high electric field. The method was already successfully applied for reconstruction of electric field distribution in agar phantoms. Before the next step towards in vivo experiments is taken, monitoring of electric field distribution during electroporation of ex vivo tissue ex vivo and feasibility for its use in electroporation based treatments needed to be evaluated. Sequences of high voltage pulses were applied to chicken liver tissue in order to expose it to electric field which was measured by means of MREIT. MREIT was also evaluated for its use in electroporation based treatments by calculating electric field distribution for two regions, the tumor and the tumor-liver region, in a numerical model based on data obtained from clinical study on electrochemotherapy treatment of deep-seated tumors. Electric field distribution inside tissue was successfully measured ex vivo using MREIT and significant changes of tissue electrical conductivity were observed in the region of the highest electric field. A good agreement was obtained between the electric field distribution obtained by MREIT and the actual electric field distribution in evaluated regions of a numerical model, suggesting that implementation of MREIT could thus enable efficient detection of areas with insufficient electric field coverage during electroporation based treatments, thus assuring the effectiveness of the treatment.

  14. Electroporation-based treatment planning for deep-seated tumors based on automatic liver segmentation of MRI images.

    Science.gov (United States)

    Pavliha, Denis; Mušič, Maja M; Serša, Gregor; Miklavčič, Damijan

    2013-01-01

    Electroporation is the phenomenon that occurs when a cell is exposed to a high electric field, which causes transient cell membrane permeabilization. A paramount electroporation-based application is electrochemotherapy, which is performed by delivering high-voltage electric pulses that enable the chemotherapeutic drug to more effectively destroy the tumor cells. Electrochemotherapy can be used for treating deep-seated metastases (e.g. in the liver, bone, brain, soft tissue) using variable-geometry long-needle electrodes. To treat deep-seated tumors, patient-specific treatment planning of the electroporation-based treatment is required. Treatment planning is based on generating a 3D model of the organ and target tissue subject to electroporation (i.e. tumor nodules). The generation of the 3D model is done by segmentation algorithms. We implemented and evaluated three automatic liver segmentation algorithms: region growing, adaptive threshold, and active contours (snakes). The algorithms were optimized using a seven-case dataset manually segmented by the radiologist as a training set, and finally validated using an additional four-case dataset that was previously not included in the optimization dataset. The presented results demonstrate that patient's medical images that were not included in the training set can be successfully segmented using our three algorithms. Besides electroporation-based treatments, these algorithms can be used in applications where automatic liver segmentation is required.

  15. Electroporation-based treatment planning for deep-seated tumors based on automatic liver segmentation of MRI images.

    Directory of Open Access Journals (Sweden)

    Denis Pavliha

    Full Text Available Electroporation is the phenomenon that occurs when a cell is exposed to a high electric field, which causes transient cell membrane permeabilization. A paramount electroporation-based application is electrochemotherapy, which is performed by delivering high-voltage electric pulses that enable the chemotherapeutic drug to more effectively destroy the tumor cells. Electrochemotherapy can be used for treating deep-seated metastases (e.g. in the liver, bone, brain, soft tissue using variable-geometry long-needle electrodes. To treat deep-seated tumors, patient-specific treatment planning of the electroporation-based treatment is required. Treatment planning is based on generating a 3D model of the organ and target tissue subject to electroporation (i.e. tumor nodules. The generation of the 3D model is done by segmentation algorithms. We implemented and evaluated three automatic liver segmentation algorithms: region growing, adaptive threshold, and active contours (snakes. The algorithms were optimized using a seven-case dataset manually segmented by the radiologist as a training set, and finally validated using an additional four-case dataset that was previously not included in the optimization dataset. The presented results demonstrate that patient's medical images that were not included in the training set can be successfully segmented using our three algorithms. Besides electroporation-based treatments, these algorithms can be used in applications where automatic liver segmentation is required.

  16. Exploration of automatic optimisation for CUDA programming

    KAUST Repository

    Al-Mouhamed, Mayez

    2014-09-16

    © 2014 Taylor & Francis. Writing optimised compute unified device architecture (CUDA) program for graphic processing units (GPUs) is complex even for experts. We present a design methodology for a restructuring tool that converts C-loops into optimised CUDA kernels based on a three-step algorithm which are loop tiling, coalesced memory access and resource optimisation. A method for finding possible loop tiling solutions with coalesced memory access is developed and a simplified algorithm for restructuring C-loops into an efficient CUDA kernel is presented. In the evaluation, we implement matrix multiply (MM), matrix transpose (M-transpose), matrix scaling (M-scaling) and matrix vector multiply (MV) using the proposed algorithm. We present the analysis of the execution time and GPU throughput for the above applications, which favourably compare to other proposals. Evaluation is carried out while scaling the problem size and running under a variety of kernel configurations. The obtained speedup is about 28-35% for M-transpose compared to NVIDIA Software Development Kit, 33% speedup for MV compared to general purpose computation on graphics processing unit compiler, and more than 80% speedup for MM and M-scaling compared to CUDA-lite.

  17. Designing Lead Optimisation of MMP-12 Inhibitors

    Directory of Open Access Journals (Sweden)

    Matteo Borrotti

    2014-01-01

    Full Text Available The design of new molecules with desired properties is in general a very difficult problem, involving heavy experimentation with high investment of resources and possible negative impact on the environment. The standard approach consists of iteration among formulation, synthesis, and testing cycles, which is a very long and laborious process. In this paper we address the so-called lead optimisation process by developing a new strategy to design experiments and modelling data, namely, the evolutionary model-based design for optimisation (EDO. This approach is developed on a very small set of experimental points, which change in relation to the response of the experimentation according to the principle of evolution and insights gained through statistical models. This new procedure is validated on a data set provided as test environment by Pickett et al. (2011, and the results are analysed and compared to the genetic algorithm optimisation (GAO as a benchmark. The very good performance of the EDO approach is shown in its capacity to uncover the optimum value using a very limited set of experimental points, avoiding unnecessary experimentation.

  18. Procedure for Application-Oriented Optimisation of Marine Propellers

    Directory of Open Access Journals (Sweden)

    Florian Vesting

    2016-11-01

    Full Text Available The use of automated optimisation in engineering applications is emerging. In particular, nature inspired algorithms are frequently used because of their variability and robust application in constraints and multi-objective optimisation problems. The purpose of this paper is the comparison of four different algorithms and several optimisation strategies on a set of seven test propellers in realistic industrial design setting. The propellers are picked from real commercial projects and the manual final designs were delivered to customers. The different approaches are evaluated and final results of the automated optimisation toolbox are compared with designs generated in a manual design process. We identify a two-stage optimisation for marine propellers, where the geometry is first modified by parametrised geometry distribution curves to gather knowledge of the test case. Here we vary the optimisation strategy in terms of applied algorithms, constraints and objectives. A second supporting optimisation aims to improve the design by locally changing the geometry, based on the results of the first optimisation. The optimisation algorithms and strategies yield propeller designs that are comparable to the manually designed propeller blade geometries, thus being suitable as robust and advanced design support tools. The supporting optimisation, with local modification of the blade geometry and the proposed cavity shape constraints, features particular good performance in modifying cavitation on the blade and is, with the AS NSGA-II (adaptive surrogate-assisted NSGA-II, superior in lead time.

  19. Intravaginal HPV DNA vaccination with electroporation induces local CD8+ T-cell immune responses and antitumor effects against cervicovaginal tumors.

    Science.gov (United States)

    Sun, Y; Peng, S; Qiu, J; Miao, J; Yang, B; Jeang, J; Hung, C-F; Wu, T-C

    2015-07-01

    Therapeutic human papillomavirus (HPV) vaccines have the potential to inhibit the progression of an established HPV infection to precancer and cancer lesions by targeting HPV oncoproteins. We have previously developed a therapeutic DNA vaccine encoding calreticulin (CRT) linked to E7, CRT/E7 DNA vaccine, for use in the treatment of HPV-associated lesions. Since the transfection efficiency of DNA vaccines administered in vivo is typically low, we examined the use of electroporation as well as different routes of administration to enhance antigen-specific tumor control. We tested the effects of the CRT/E7 DNA vaccine administered intramuscularly or intravaginally, with or without electroporation, on the generation of CD8+ T-cell immunity and therapeutic antitumor effects in HPV16 E7-expressing cervicovaginal tumor-bearing mice. We found that intravaginal vaccination of CRT/E7 DNA followed by electroporation-induced potent E7-specific CD8(+) T-cell responses in the cervicovaginal tract, compared with intramuscular injection followed by electroporation. Furthermore, tumor-bearing mice vaccinated intravaginally followed by electroporation had an enhanced survival, antitumor effects and local production of IFN-γ+CD8+ T cells compared with those vaccinated intramuscularly with electroporation. Thus, we show that intravaginal CRT/E7 DNA vaccination followed by electroporation generates the most potent therapeutic antitumor effects against an orthotopic E7-expressing tumor model. The current study will have significant clinical implications once a clinically applicable electroporation device for intravaginal use becomes available.

  20. Railway vehicle performance optimisation using virtual homologation

    Science.gov (United States)

    Magalhães, H.; Madeira, J. F. A.; Ambrósio, J.; Pombo, J.

    2016-09-01

    Unlike regular automotive vehicles, which are designed to travel in different types of roads, railway vehicles travel mostly in the same route during their life cycle. To accept the operation of a railway vehicle in a particular network, a homologation process is required according to local standard regulations. In Europe, the standards EN 14363 and UIC 518, which are used for railway vehicle acceptance, require on-track tests and/or numerical simulations. An important advantage of using virtual homologation is the reduction of the high costs associated with on-track tests by studying the railway vehicle performance in different operation conditions. This work proposes a methodology for the improvement of railway vehicle design with the objective of its operation in selected railway tracks by using optimisation. The analyses required for the vehicle improvement are performed under control of the optimisation method global and local optimisation using direct search. To quantify the performance of the vehicle, a new objective function is proposed, which includes: a Dynamic Performance Index, defined as a weighted sum of the indices obtained from the virtual homologation process; the non-compensated acceleration, which is related to the operational velocity; and a penalty associated with cases where the vehicle presents an unacceptable dynamic behaviour according to the standards. Thus, the optimisation process intends not only to improve the quality of the vehicle in terms of running safety and ride quality, but also to increase the vehicle availability via the reduction of the time for a journey while ensuring its operational acceptance under the standards. The design variables include the suspension characteristics and the operational velocity of the vehicle, which are allowed to vary in an acceptable range of variation. The results of the optimisation lead to a global minimum of the objective function in which the suspensions characteristics of the vehicle are

  1. Electroporation-aided DNA immunization generates polyclonal antibodies against the native conformation of human endothelin B receptor.

    Science.gov (United States)

    Allard, Bertrand; Priam, Fabienne; Deshayes, Frédérique; Ducancel, Frédéric; Boquet, Didier; Wijkhuisen, Anne; Couraud, Jean-Yves

    2011-09-01

    Endothelin B receptor (ET(B)R) is a G protein-coupled receptor (GPCR) specific for endothelin peptides (including endothelin-1, ET1), which mediates a variety of key physiological functions in normal tissues, such as modulation of vasomotor tone, tissue differentiation, or cell proliferation. Moreover, ET(B)R, overexpressed in various cancer cells including melanoma, has been implicated in the growth and progression of tumors, as well as in controlling T cell homing to tumors. To gather information on receptor structure and function, antibodies are generally considered choice molecular probes, but generation of such reagents against the native conformation of GPCRs is a real technical challenge. Here, we show that electroporation-aided genetic immunization, coupled to cardiotoxin pretreatment, is a simple and very efficient method to raise large amounts of polyclonal antibodies highly specific for native human ET(B)R (hET(B)R), as assessed by both flow cytometry analysis of different stably transfected cell lines and a new and rapid cell-based enzyme-linked immunosorbent assay that we also describe. The antibodies recognized two major epitopes on hET(B)R, mapped within the N-terminal extracellular domain. They were used to reveal hET(B)R on membranes of three different human melanoma cell lines, by flow cytometry and confocal microscopy, a method that we show is more relevant than mRNA polymerase chain reaction in assessing receptor expression. In addition, ET-1 partially competed with antibodies for receptor binding. The strategy described here, thus, efficiently generated new immunological tools to further analyze the role of ET(B)R under both normal and pathological conditions, including cancers. Above all, it can now be used to raise monoclonal antibodies against hET(B)R and, more generally, against GPCRs that constitute, by far, the largest reservoir of potential pharmacological targets.

  2. Automatic optimisation of gamma dose rate sensor networks: The DETECT Optimisation Tool

    DEFF Research Database (Denmark)

    Helle, K.B.; Müller, T.O.; Astrup, Poul;

    2014-01-01

    chosen using regular grids or according to administrative constraints. Nowadays, however, the choice can be based on more realistic risk assessment, as it is possible to simulate potential radioactive plumes. To support sensor planning, we developed the DETECT Optimisation Tool (DOT) within the scope...... monitoring network for early detection of radioactive plumes or for the creation of dose maps. The DOT is implemented as a stand-alone easy-to-use JAVA-based application with a graphical user interface and an R backend. Users can run evaluations and optimisations, and display, store and download the results...

  3. Improved electroporation parameters of delivering silver nanoparticles into living C666 cells for surface-enhanced Raman scattering

    Energy Technology Data Exchange (ETDEWEB)

    Yu Yun; Lin Juqiang; Huang Zufang; Xi Gangqin; Lin Duo; Chen Yongjian; Chen Rong [Key Laboratory of Optoelectronic Science and Technology for Medicine, Ministry of Education, Fujian Normal University, Fuzhou 350007 (China); Zeng Haishan, E-mail: chenr@fjnu.edu.cn [Cancer Imaging Department, British Columbia Cancer Research Centre, Vancouver, B.C., V5Z 1L3 (Canada)

    2011-01-01

    Electroporation assisted metallic nanoparticle delivery has been shown by our previous work to significantly reduce the time of sample preparation for surface-enhanced Raman spectroscopy (SERS) measurements of biological cells. In this research note, we report our experimental work to optimize the electroporation parameters, including adjustment of the pulse pattern and operation temperature, for fastest delivery of silver nanoparticles into living C666 cells (a human nasopharyngeal carcinoma cell line). The delivery efficiency was evaluated by the integrated intensity of whole cell SERS spectrum. Our work concluded that the silver nanoparticle delivery rate is best under the electroporation condition of using 4 consecutive 350 V (875 V/cm) rectangular electric pulses of 1 ms, 10 ms, 10 ms, and 1 ms durations respectively. Low temperature (0{approx}4 deg. C) is necessary for improving the delivery efficiency of silver nanoparticles.

  4. An optimized electroporation method for delivering nanoparticles into living cells for surface-enhanced Raman scattering imaging

    Science.gov (United States)

    Yu, Yun; Wang, Jing; Lin, Juqiang; Lin, Duo; Chen, Weiwei; Feng, Shangyuan; Huang, Zufang; Li, Yongzeng; Huang, Hao; Shi, Hong; Chen, Rong

    2016-04-01

    The existing electroporation method can rapidly deliver nanoparticles (NPs) into living cells for intracellular surface-enhanced Raman scattering (SERS) imaging. Unfortunately, the cellular SERS signals are major from molecules located near the two poles of the cell facing toward to the electrodes because most NPs enter cells through these two poles and easily happen to aggregate there. Here, we present an optimized electroporation method for transferring NPs into living cells to obtain a uniform NPs distribution. The distribution of intracellular NPs was monitored by the SERS signal of 4-mercaptobenzoic acid, which is sandwiched between the Au-Ag core-shell and validated by TEM images. In addition, based on this uniform distribution of NPs, we then detected the distribution of cellular molecules like phenylalanine and lipid via SERS imaging. Results demonstrate the great potential for the optimized electroporation-based SERS imaging in cellular study.

  5. Real-time optimisation of the Hoa Binh reservoir, Vietnam

    DEFF Research Database (Denmark)

    Richaud, Bertrand; Madsen, Henrik; Rosbjerg, Dan

    2011-01-01

    -time optimisation. First, the simulation-optimisation framework is applied for optimising reservoir operating rules. Secondly, real-time and forecast information is used for on-line optimisation that focuses on short-term goals, such as flood control or hydropower generation, without compromising the deviation......Multi-purpose reservoirs often have to be managed according to conflicting objectives, which requires efficient tools for trading-off the objectives. This paper proposes a multi-objective simulation-optimisation approach that couples off-line rule curve optimisation with on-line real...... of the forecast is addressed. The results illustrate the importance of a sufficient forecast lead time to start pre-releasing water in flood situations....

  6. Methods for Optimisation of the Laser Cutting Process

    DEFF Research Database (Denmark)

    Dragsted, Birgitte

    This thesis deals with the adaptation and implementation of various optimisation methods, in the field of experimental design, for the laser cutting process. The problem in optimising the laser cutting process has been defined and a structure for at Decision Support System (DSS......) for the optimisation of the laser cutting process has been suggested. The DSS consists of a database with the currently used and old parameter settings. Also one of the optimisation methods has been implemented in the DSS in order to facilitate the optimisation procedure for the laser operator. The Simplex Method has...... been adapted in two versions. A qualitative one, that by comparing the laser cut items optimise the process and a quantitative one that uses a weighted quality response in order to achieve a satisfactory quality and after that maximises the cutting speed thus increasing the productivity of the process...

  7. High-throughput and real-time study of single cell electroporation using microfluidics: effects of medium osmolarity.

    Science.gov (United States)

    Wang, Hsiang-Yu; Lu, Chang

    2006-12-20

    Electroporation has been widely accepted as an important tool for the delivery of exogenous molecules into cells. Previous mechanistic studies have been carried out by observing either the average behavior from a large population of cells or the response from a small number of single cells. In this study, we demonstrated a novel microfluidic method with high throughput (up to 30 Hz) for real-time studies of single cell electroporation events. Electroporation occurred when cells flowed through a section of a microfluidic channel defined by special geometry. A CCD camera was used to monitor the response of cells starting from the onset of the electroporation. We studied the swelling of Chinese hamster ovary cells and the rupture of cell membrane during electroporation using this technique. We applied buffers with different osmolarities to investigate the effects of medium osmolarity, based on results from a population of single cells. We were able to establish the distributions of the rates of swelling and membrane rupture in the cell population. We also explored establishing the correlation between the property (the cell diameter) and the behavior (the swelling rate) of single cells. Our results indicated that the processes of swelling and rupture occurred more rapidly in the hypotonic or hypertonic buffers than in the isotonic buffer. Statistical analysis did not reveal strong linear correlation between the cell size and the swelling rate. These proof-of-concept studies reveal the potential of applying microfluidics to study electroporation of a cell population at single cell level in real time with high throughput. The limitations associated with this approach were also addressed.

  8. Highly efficient transformation of intact yeast-like conidium cells of Tremella fuciformin by electroporation

    Institute of Scientific and Technical Information of China (English)

    GUO LiQiong; LIU Yong; ZHAO ShuXian; LIU ErXian; LIU JunFang

    2008-01-01

    Tremella fuciformis is one of higher basidiomycetes. Its basidiospore can reproduce yeast-like conidia, also called the blastospore by budding. The yeast-like conidia of T. Fuciformis is monokaryotic and easy to culture by submerged fermentation similar to yeast. So it is a good recipient cell for exogenous gone expression. In this study, two expression vectors pGIg-gfp containing gpd-GI promoter and gfp gone and pGIg-hph containing gpd-GI promoter and hph gone were constructed. The lowest sensitive concentration of hygromycin for the blastospore was determined on three types of media. Our ex-perimenta showed that the lowest sensitive concentration of hygromycin for the blastospore was 5 μg/mL on MA medium. The intact blastospores were transformed with the expression vector pGIg-hph by electroporation. The putative transformants were obtained by the MA selective medium. Experi-mental results showed that the most effective parameters for the electroporation of intact blastospores were obtained by using STM buffer, 1.0×108 cells/mL of blastospores, 200 μL in transformation volume, 6 μg plasmid, 2.0 kV/cm of electric pulse voltage, stillness culturing on MB liquid medium for 48 h after electroporation. In these transformation conditions, the efficiency reached 277 colonies/μg DNA. With the optimal parameters. The putative co-transformants were obtained by the MA selective medium. Eight randomly selected colonies from the vast putative co-transformants were analyzed by PCR de-tection and Southern blotting. The experiments showed that the gfp was integrated into the genomes of three transformants. The co-transformation efficiency was 37.5%. Green fluorescence was observed under laser scanning confocal microscope in these gfp positive transformants. This indicates that the exogenous gfp can be expressed effectively in the yeast-like conidia of T. Fuciformis.

  9. Gene therapy by electroporation for the treatment of chronic renal failure in companion animals

    Directory of Open Access Journals (Sweden)

    Pope Melissa A

    2009-01-01

    Full Text Available Abstract Background Growth hormone-releasing hormone (GHRH plasmid-based therapy for the treatment of chronic renal failure and its complications was examined. Companion dogs (13.1 ± 0.8 years, 29.4 ± 5.01 kg and cats (13.2 ± 0.9 years, 8.5 ± 0.37 kg received a single 0.4 mg or 0.1 mg species-specific plasmid injection, respectively, intramuscularly followed by electroporation, and analyzed up to 75 days post-treatment; controls underwent electroporation without plasmid administration. Results Plasmid-treated animals showed an increase in body weight (dogs 22.5% and cats 3.2% compared to control animals, and displayed improved quality of life parameters including significant increases in appetite, activity, mentation and exercise tolerance levels. Insulin-like growth factor I (IGF-I, the downstream effector of GHRH levels were increased in the plasmid treated animals. Hematological parameters were also significantly improved. Protein metabolism changes were observed suggesting a shift from a catabolic to an anabolic state in the treated animals. Blood urea nitrogen and creatinine did not show any significant changes suggesting maintenance of kidney function whereas the control animal's renal function deteriorated. Treated animals survived longer than control animals with 70% of dogs and 80% of cats surviving until study day 75. Only 17% and 40% of the control dogs and cats, respectively, survived to day 75. Conclusion Improved quality of life, survival and general well-being indicate that further investigation is warranted, and show the potential of a plasmid-based therapy by electroporation in preventing and managing complications of renal insufficiency.

  10. A theoretical study of single-cell electroporation in a microchannel.

    Science.gov (United States)

    Movahed, Saeid; Li, Dongqing

    2013-02-01

    Electroporation of a single cell in a microchannel was studied. The effects of electrical (e.g., strength of the electric pulse) and geometrical (e.g., microchannel height, electrode size and position) parameters on cell membrane permeabilization were investigated. The electrodes were assumed to be embedded in the walls of the microchannel; the cell was suspended between these two electrodes. By keeping the electric pulse constant, increasing the microchannel height reduces the number and the radius of the biggest nanopores, as well as the electroporated area of the cell membrane. If the width of the electrodes is bigger than the cell diameter, the transmembrane potential will be centralized and have a sinusoidal distribution around the cell if nanopores are not generated. As the width of the electrode decreases and becomes smaller than the cell diameter, the local transmembrane potential decreases; in the nonelectroporative area, the transmembrane potential distribution deviates from the sinusoidal behavior; the induced transmembrane potential also concentrates around the poles of the cell membrane (the nearest points of the cell membrane to the electrodes). During cell membrane permeabilization, the biggest nanopores are initially created at the poles and then the nanopore population expands toward the equator. The number of the created nanopores reaches its maximal value within a few microseconds; further presence of the electric pulse may not influence the number and location of the created nanopores anymore but will develop the generated nanopores. Strengthening the electric pulse intensifies the size and number of the created nanopores as well as the electroporated area on the cell membrane.

  11. Design Optimisation and Conrol of a Pilot Operated Seat Valve

    DEFF Research Database (Denmark)

    Nielsen, Brian; Andersen, Torben Ole; Hansen, Michael Rygaard

    2004-01-01

    The paper gives an approach for optimisation of the bandwidth of a pilot operated seat valve for mobile applications. Physical dimensions as well as parameters of the implemented control loop are optimised simultaneously. The frequency response of the valve varies as a function of the pressure drop...... across the valve, and it is found to be necessary to scale the controller parameters in the optimised design as a function of pressure drop....

  12. Mechatronic System Design Based On An Optimisation Approach

    DEFF Research Database (Denmark)

    Andersen, Torben Ole; Pedersen, Henrik Clemmensen; Hansen, Michael Rygaard

    The envisaged objective of this paper project is to extend the current state of the art regarding the design of complex mechatronic systems utilizing an optimisation approach. We propose to investigate a novel framework for mechatronic system design. The novelty and originality being the use...... of optimisation techniques. The methods used to optimise/design within the classical disciplines will be identified and extended to mechatronic system design....

  13. Percutaneous Irreversible Electroporation of Unresectable Hilar Cholangiocarcinoma (Klatskin Tumor): A Case Report

    Energy Technology Data Exchange (ETDEWEB)

    Melenhorst, Marleen C. A. M., E-mail: m.melenhorst@vumc.nl; Scheffer, Hester J., E-mail: hj.scheffer@vumc.nl; Vroomen, Laurien G. P. H., E-mail: la.vroomen@vumc.nl [VU University Medical Center, Department of Radiology and Nuclear Medicine (Netherlands); Kazemier, Geert, E-mail: g.kazemier@vumc.nl; Tol, M. Petrousjka van den, E-mail: mp.vandentol@vumc.nl [VU University Medical Center, Department of Surgery (Netherlands); Meijerink, Martijn R., E-mail: mr.meijerink@vumc.nl [VU University Medical Center, Department of Radiology and Nuclear Medicine (Netherlands)

    2016-01-15

    Irreversible electroporation (IRE) is a novel image-guided ablation technique that is rapidly gaining popularity in the treatment of malignant tumors located near large vessels or bile ducts. The presence of metal objects in the ablation zone, such as Wallstents, is generally considered a contraindication for IRE, because tissue heating due to power conduction may lead to thermal complications. This report describes a 66-year-old female with a Bismuth–Corlette stage IV unresectable cholangiocarcinoma with a metallic Wallstent in the common bile duct, who was safely treated with percutaneous IRE with no signs for relapse 1 year after the procedure.

  14. Percutaneous Irreversible Electroporation of Unresectable Hilar Cholangiocarcinoma (Klatskin Tumor): A Case Report.

    Science.gov (United States)

    Melenhorst, Marleen C A M; Scheffer, Hester J; Vroomen, Laurien G P H; Kazemier, Geert; van den Tol, M Petrousjka; Meijerink, Martijn R

    2016-01-01

    Irreversible electroporation (IRE) is a novel image-guided ablation technique that is rapidly gaining popularity in the treatment of malignant tumors located near large vessels or bile ducts. The presence of metal objects in the ablation zone, such as Wallstents, is generally considered a contraindication for IRE, because tissue heating due to power conduction may lead to thermal complications. This report describes a 66-year-old female with a Bismuth-Corlette stage IV unresectable cholangiocarcinoma with a metallic Wallstent in the common bile duct, who was safely treated with percutaneous IRE with no signs for relapse 1 year after the procedure.

  15. Highly efficient transformation of intact yeast-like conidium cells of Tremella fuciformis by electroporation

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Tremella fuciformis is one of higher basidiomycetes. Its basidiospore can reproduce yeast-like conidia, also called the blastospore by budding. The yeast-like conidia of T. fuciformis is monokaryotic and easy to culture by submerged fermentation similar to yeast. So it is a good recipient cell for exogenous gene expression. In this study, two expression vectors pGlg-gfp containing gpd-Gl promoter and gfp gene and pGlg-hph containing gpd-Gl promoter and hph gene were constructed. The lowest sensitive concentration of hygromycin for the blastospore was determined on three types of media. Our ex- periments showed that the lowest sensitive concentration of hygromycin for the blastospore was 5 μg/mL on MA medium. The intact blastospores were transformed with the expression vector pGlg-hph by electroporation. The putative transformants were obtained by the MA selective medium. Experi- mental results showed that the most effective parameters for the electroporation of intact blastospores were obtained by using STM buffer, 1.0×108 cells/mL of blastospores, 200 μL in transformation volume, 6 μg plasmid, 2.0 kV/cm of electric pulse voltage, stillness culturing on MB liquid medium for 48 h after electroporation. In these transformation conditions, the efficiency reached 277 colonies/μg DNA. Co-transformation of plasmid pGlg-gfp and pGlg-hph with ratio of 1:1 was performed by electroporation with the optimal parameters. The putative co-transformants were obtained by the MA selective medium. Eight randomly selected colonies from the vast putative co-transformants were analyzed by PCR de- tection and Southern blotting. The experiments showed that the gfp was integrated into the genomes of three transformants. The co-transformation efficiency was 37.5%. Green fluorescence was observed under laser scanning confocal microscope in these gfp positive transformants. This indicates that the exogenous gfp can be expressed effectively in the yeast-like conidia of T. fuciformis.

  16. Comparison of Different Electroporation Parameters on Transfection Efficiency of Sheep Testicular Cells

    Directory of Open Access Journals (Sweden)

    Sarah Niakanrisi

    2016-09-01

    Full Text Available Objective: Electroporation can be a highly efficient method for introducing the foreign genetic materials into the targeted cells for transient and/or permanent genetic modification. Considering the application of this technique as a very efficient method for drug, oligonucleotide, antibody and plasmid delivery for clinical applications and production of transgenic animals, the present study aimed to optimize the transfection efficiency of sheep testicular cells including spermatogonial stem cells (SSCs via electroporation. Materials and Methods: This study is an experimental research conducted in Biotechnology Research Center (Avicenna Research Institute, Tehran, Iran from September 2013 to March 2014. Following isolation and propagation of one-month lamb testicular cells (SSCs and somatic testicular cells including; Sertoli, Leydig, and myoid cells, the effect of different electroporation parameters including total voltages (280, 320, and 350 V, burst durations (10, 8, and 5 milliseconds, burst modes (single or double and addition of dimethyl sulfoxide (DMSO were evaluated on transfection efficiency, viability rate and mean fluorescent intensity (MFI of sheep testicular cells. Results: The most transfection efficiency was obtained in 320 V/8 milliseconds/single burst group in transduction medium with and without DMSO. There was a significantly inverse correlation between transfection efficiency with application of both following parameters: addition of DMSO and double burst. After transfection, the highest and lowest viability rates of testicular cells were demonstrated in 320 V/8 milliseconds with transduction medium without DMSO and 350 V/5 milliseconds in medium containing DMSO. Addition of DMSO to transduction medium in all groups significantly decreased the viability rate. The comparison of gene expression indicated that Sertoli and SSCs had the most fluorescence intensity in 320 V/double burst/DMSO positive. However, myoid and Leydig

  17. Educational application for visualization and analysis of electric field strength in multiple electrode electroporation

    Directory of Open Access Journals (Sweden)

    Mahnič-Kalamiza Samo

    2012-10-01

    Full Text Available Abstract Background Electrochemotherapy is a local treatment that utilizes electric pulses in order to achieve local increase in cytotoxicity of some anticancer drugs. The success of this treatment is highly dependent on parameters such as tissue electrical properties, applied voltages and spatial relations in placement of electrodes that are used to establish a cell-permeabilizing electric field in target tissue. Non-thermal irreversible electroporation techniques for ablation of tissue depend similarly on these parameters. In the treatment planning stage, if oversimplified approximations for evaluation of electric field are used, such as U/d (voltage-to-distance ratio, sufficient field strength may not be reached within the entire target (tumor area, potentially resulting in treatment failure. Results In order to provide an aid in education of medical personnel performing electrochemotherapy and non-thermal irreversible electroporation for tissue ablation, assist in visualizing the electric field in needle electrode electroporation and the effects of changes in electrode placement, an application has been developed both as a desktop- and a web-based solution. It enables users to position up to twelve electrodes in a plane of adjustable dimensions representing a two-dimensional slice of tissue. By means of manipulation of electrode placement, i.e. repositioning, and the changes in electrical parameters, the users interact with the system and observe the resulting electrical field strength established by the inserted electrodes in real time. The field strength is calculated and visualized online and instantaneously reflects the desired changes, dramatically improving the user friendliness and educational value, especially compared to approaches utilizing general-purpose numerical modeling software, such as finite element modeling packages. Conclusion In this paper we outline the need and offer a solution in medical education in the field of

  18. Synthesis of ABA Tri-Block Co-Polymer Magnetopolymersomes via Electroporation for Potential Medical Application

    Directory of Open Access Journals (Sweden)

    Jennifer Bain

    2015-12-01

    Full Text Available The ABA tri-block copolymer poly(2-methyloxazoline–poly(dimethylsiloxane–poly(2-methyloxazoline (PMOXA–PDMS–PMOXA is known for its capacity to mimic a bilayer membrane in that it is able to form vesicular polymersome structures. For this reason, it is the subject of extensive research and enables the development of more robust, adaptable and biocompatible alternatives to natural liposomes for biomedical applications. However, the poor solubility of this polymer renders published methods for forming vesicles unreproducible, hindering research and development of these polymersomes. Here we present an adapted, simpler method for the production of PMOXA–PDMS–PMOXA polymersomes of a narrow polydispersity (45 ± 5.8 nm, via slow addition of aqueous solution to a new solvent/polymer mixture. We then magnetically functionalise these polymersomes to form magnetopolymersomes via in situ precipitation of iron-oxide magnetic nanoparticles (MNPs within the PMOXA–PDMS–PMOXA polymersome core and membrane. This is achieved using electroporation to open pores within the membrane and to activate the formation of MNPs. The thick PMOXA–PDMS–PMOXA membrane is well known to be relatively non-permeable when compared to more commonly used di-block polymer membranes due a distinct difference in both size and chemistry and therefore very difficult to penetrate using standard biological methods. This paper presents for the first time the application of electroporation to an ABA tri-block polymersome membrane (PMOXA–PDMS–PMOXA for intravesicular in situ precipitation of uniform MNPs (2.6 ± 0.5 nm. The electroporation process facilitates the transport of MNP reactants across the membrane yielding in situ precipitation of MNPs. Further to differences in length and chemistry, a tri-block polymersome membrane structure differs from a natural lipid or di-block polymer membrane and as such the application and effects of electroporation on this type of

  19. Long-term effectiveness of irreversible electroporation in a murine model of colorectal liver metastasis

    OpenAIRE

    Ivorra Cano, Antoni; Sánchez Velázquez, Patricia; Castellví, Quim; Villanueva, Alberto; Iglesias Coma, Mar; Quesada Diez, Rita; Pañella-Vilamú, Clara; Cáceres Aguilar, Mario; Dorcaratto, Dimitri; Andaluz, Anna; Moll, Xavier; Burdío, José Miguel; Grande Posa, Luís; Burdío Pinilla, Fernando

    2017-01-01

    Irreversible electroporation (IRE) has recently gained in popularity as an ablative technique, however little is known about its oncological long-term outcomes. To determine the long-time survival of animals treated with a high dose of IRE and which histological changes it induces in tumoral tissue, IRE ablation was performed in forty-six athymic-nude mice with KM12C tumors implanted in the liver by applying electric current with different voltages (2000 V/cm, 1000 V/cm). The tumors were allo...

  20. Measurement of the permeability and resealing time constant of the electroporated mammalian cell membranes

    Energy Technology Data Exchange (ETDEWEB)

    Shirakashi, Ryo [Tokyo Univ., Inst. of Industrial Science, Tokyo (Japan); Sukhorukov, Vladimir L.; Zimmermann, Ulrich [Wuerzburg Univ. Biozentrum, Lehrstuhl fuer Biotechnologie, Wuerzburg (Germany); Tanasawa, Ichiro [Nihon Univ., Dept. of Mechanical Engineering, Koriyama (Japan)

    2004-10-01

    In this study a new method is presented for measuring the transient permeability of mammalian cell membranes to sugar and electrolyte molecules based on the volumetric response of cells subjected to electroporation. The time constant of membrane resealing was determined independently by flow cytometry using a fluorescent dye as the reporter molecule. The volumetric and dye uptake data were analyzed with a model relating the cell volume changes to the solute transport across the reversibly permeabilized cell membrane. The experimental approach developed here might be useful for estimating the amount of electroinjected molecules, which are difficult to measure directly. (Author)

  1. Skin electroporation: effects on transgene expression, DNA persistence and local tissue environment

    DEFF Research Database (Denmark)

    Roos, Anna-Karin; Eriksson, Fredrik; Timmons, James A

    2009-01-01

    of vaccines against both infectious diseases and cancer. In vivo electrovaccination (gene delivery followed by electroporation) is currently being investigated in several clinical trials, including DNA delivery to healthy volunteers. However, the mode of action at molecular level is not yet fully understood....... METHODOLOGY/PRINCIPAL FINDINGS: This study investigates intradermal DNA electrovaccination in detail and describes the effects on expression of the vaccine antigen, plasmid persistence and the local tissue environment. Gene profiling of the vaccination site showed that the combination of DNA...

  2. Improving Vector Evaluated Particle Swarm Optimisation by incorporating nondominated solutions.

    Science.gov (United States)

    Lim, Kian Sheng; Ibrahim, Zuwairie; Buyamin, Salinda; Ahmad, Anita; Naim, Faradila; Ghazali, Kamarul Hawari; Mokhtar, Norrima

    2013-01-01

    The Vector Evaluated Particle Swarm Optimisation algorithm is widely used to solve multiobjective optimisation problems. This algorithm optimises one objective using a swarm of particles where their movements are guided by the best solution found by another swarm. However, the best solution of a swarm is only updated when a newly generated solution has better fitness than the best solution at the objective function optimised by that swarm, yielding poor solutions for the multiobjective optimisation problems. Thus, an improved Vector Evaluated Particle Swarm Optimisation algorithm is introduced by incorporating the nondominated solutions as the guidance for a swarm rather than using the best solution from another swarm. In this paper, the performance of improved Vector Evaluated Particle Swarm Optimisation algorithm is investigated using performance measures such as the number of nondominated solutions found, the generational distance, the spread, and the hypervolume. The results suggest that the improved Vector Evaluated Particle Swarm Optimisation algorithm has impressive performance compared with the conventional Vector Evaluated Particle Swarm Optimisation algorithm.

  3. Robust Optimisation Approach for Vehicle Routing Problems with Uncertainty

    Directory of Open Access Journals (Sweden)

    Liang Sun

    2015-01-01

    Full Text Available We formulated a solution procedure for vehicle routing problems with uncertainty (VRPU for short with regard to future demand and transportation cost. Unlike E-SDROA (expectation semideviation robust optimisation approach for solving the proposed problem, the formulation focuses on robust optimisation considering situations possibly related to bidding and capital budgets. Besides, numerical experiments showed significant increments in the robustness of the solutions without much loss in solution quality. The differences and similarities of the robust optimisation model and existing robust optimisation approaches were also compared.

  4. Biorefinery plant design, engineering and process optimisation

    DEFF Research Database (Denmark)

    Holm-Nielsen, Jens Bo; Ehimen, Ehiazesebhor Augustine

    2014-01-01

    applicable for the planning and upgrading of intended biorefinery systems, and includes discussions on the operation of an existing lignocellulosic-based biorefinery platform. Furthermore, technical considerations and tools (i.e., process analytical tools) which could be applied to optimise the operations......Before new biorefinery systems can be implemented, or the modification of existing single product biomass processing units into biorefineries can be carried out, proper planning of the intended biorefinery scheme must be performed initially. This chapter outlines design and synthesis approaches...

  5. SIROCCO. Silent rotors by acoustic optimisation

    Energy Technology Data Exchange (ETDEWEB)

    Schepers, J.G.; Curvers, A. [ECN Wind Energy, Petten (Netherlands); Oerlemans, S. [National Aerospace Laboratory NLR, Amsterdam (Netherlands); Braun, K.; Lutz, T.; Herrig, A.; Wuerz, W. [University of Stuttgart, Stuttgart (Germany); Matesanz, A.; Garcillan, L. [Gamesa Eolica, Madrid (Spain); Fisher, M.; Koegler, K.; Maeder, T. [GE Wind Energy/GE Global Research (United States)

    2007-07-15

    In this paper the results from the European 5th Framework project 'SIROCCO' are described. The project started in January 2003 and will end in August 2007. The main aim of the SIROCCO project is to reduce wind-turbine aerodynamic noise significantly while maintaining the aerodynamic performance. This is achieved by designing new acoustically and aerodynamically optimised airfoils for the outer part of the blade. The project focussed primarily on reducing trailing edge noise, which was broadly believed to be the dominant noise mechanism of modern wind turbines.

  6. Comparison of membrane electroporation and protein denature in response to pulsed electric field with different durations.

    Science.gov (United States)

    Huang, Feiran; Fang, Zhihui; Mast, Jason; Chen, Wei

    2013-05-01

    In this paper, we compared the minimum potential differences in the electroporation of membrane lipid bilayers and the denaturation of membrane proteins in response to an intensive pulsed electric field with various pulse durations. Single skeletal muscle fibers were exposed to a pulsed external electric field. The field-induced changes in the membrane integrity (leakage current) and the Na channel currents were monitored to identify the minimum electric field needed to damage the membrane lipid bilayer and the membrane proteins, respectively. We found that in response to a relatively long pulsed electric shock (longer than the membrane intrinsic time constant), a lower membrane potential was needed to electroporate the cell membrane than for denaturing the membrane proteins, while for a short pulse a higher membrane potential was needed. In other words, phospholipid bilayers are more sensitive to the electric field than the membrane proteins for a long pulsed shock, while for a short pulse the proteins become more vulnerable. We can predict that for a short or ultrashort pulsed electric shock, the minimum membrane potential required to start to denature the protein functions in the cell plasma membrane is lower than that which starts to reduce the membrane integrity.

  7. Static electricity powered copper oxide nanowire microbicidal electroporation for water disinfection.

    Science.gov (United States)

    Liu, Chong; Xie, Xing; Zhao, Wenting; Yao, Jie; Kong, Desheng; Boehm, Alexandria B; Cui, Yi

    2014-10-08

    Safe water scarcity occurs mostly in developing regions that also suffer from energy shortages and infrastructure deficiencies. Low-cost and energy-efficient water disinfection methods have the potential to make great impacts on people in these regions. At the present time, most water disinfection methods being promoted to households in developing countries are aqueous chemical-reaction-based or filtration-based. Incorporating nanomaterials into these existing disinfection methods could improve the performance; however, the high cost of material synthesis and recovery as well as fouling and slow treatment speed is still limiting their application. Here, we demonstrate a novel flow device that enables fast water disinfection using one-dimensional copper oxide nanowire (CuONW) assisted electroporation powered by static electricity. Electroporation relies on a strong electric field to break down microorganism membranes and only consumes a very small amount of energy. Static electricity as the power source can be generated by an individual person's motion in a facile and low-cost manner, which ensures its application anywhere in the world. The CuONWs used were synthesized through a scalable one-step air oxidation of low-cost copper mesh. With a single filtration, we achieved complete disinfection of bacteria and viruses in both raw tap and lake water with a high flow rate of 3000 L/(h·m(2)), equivalent to only 1 s of contact time. Copper leaching from the nanowire mesh was minimal.

  8. The effect of temperature and bacterial growth phase on protein extraction by means of electroporation.

    Science.gov (United States)

    Haberl-Meglič, Saša; Levičnik, Eva; Luengo, Elisa; Raso, Javier; Miklavčič, Damijan

    2016-12-01

    Different chemical and physical methods are used for extraction of proteins from bacteria, which are used in variety of fields. But on a large scale, many methods have severe drawbacks. Recently, extraction by means of electroporation showed a great potential to quickly obtain proteins from bacteria. Since many parameters are affecting the yield of extracted proteins, our aim was to investigate the effect of temperature and bacterial growth phase on the yield of extracted proteins. At the same time bacterial viability was tested. Our results showed that the temperature has a great effect on protein extraction, the best temperature post treatment being 4°C. No effect on bacterial viability was observed for all temperatures tested. Also bacterial growth phase did not affect the yield of extracted proteins or bacterial viability. Nevertheless, further experiments may need to be performed to confirm this observation, since only one incubation temperature (4°C) and one incubation time before and after electroporation (0.5 and 1h) were tested for bacterial growth phase. Based on our results we conclude that temperature is a key element for bacterial membrane to stay in a permeabilized state, so more proteins flow out of bacteria into surrounding media.

  9. Gene targeting in a HUES line of human embryonic stem cells via electroporation.

    Science.gov (United States)

    Ruby, Katherine M; Zheng, Binhai

    2009-07-01

    Genetic modification is critical for achieving the full potential of human embryonic stem (ES) cells as a tool for therapeutic development and for basic research. Targeted modifications in human ES cells have met with limited success because of the unique culture conditions for many human ES cell lines. The HUES lines of human ES cells were developed for ease of manipulation and are gaining increased utility in stem cell research. We tested conditions for gene targeting via electroporation in the HUES-9 human ES cell line and demonstrate here successful gene targeting at the gene encoding Fezf2 (also known as Fezl), a transcription factor involved in corticospinal neuron development. With a targeting strategy involving positive and negative selection that is applicable to all genes, we observed a gene targeting frequency of approximately 1.5% for Fezf2, a gene not expressed in human ES cells. We found that conditions developed for gene targeting in mouse ES cells can be readily adapted to HUES cells with few key modifications. HUES-9 cells exhibit an intrinsically high efficiency of clonal expansion and sustain electroporation-based gene targeting procedures without any significant loss of pluripotency marker expression or karyotypic stability. Thus, human ES cell lines adapted for enzymatic passage and efficient clonal expansion can be highly amenable to genetic modifications, which will facilitate their application in basic science and clinical development.

  10. In vivo non-thermal irreversible electroporation impact on rat liver galvanic apparent internal resistance

    Science.gov (United States)

    Golberg, A.; Laufer, S.; Rabinowitch, H. D.; Rubinsky, B.

    2011-02-01

    Non-thermal irreversible electroporation (NTIRE) is a biophysical phenomenon which involves application of electric field pulses to cells or tissues, causing certain rearrangements in the membrane structure leading to cell death. The treated tissue ac impedance changes induced by electroporation were shown to be the indicators for NTIRE efficiency. In a previous study we characterized in vitro tissue galvanic apparent internal resistance (GAIR) changes due to NTIRE. Here we describe an in vivo study in which we monitored the GAIR changes of a rat liver treated by NTIRE. Electrical pulses were delivered through the same Zn/Cu electrodes by which GAIR was measured. GAIR was measured before and for 3 h after the treatment at 15 min intervals. The results were compared to the established ac bioimpedance measurement method. A decrease of 33% was measured immediately after the NTIRE treatment and a 40% decrease was measured after 3 h in GAIR values; in the same time 40% and 47% decrease respectively were measured by ac bioimpedance analyses. The temperature increase due to the NTIRE was only 0.5 °C. The results open the way for an inexpensive, self-powered in vivo real-time NTIRE effectiveness measurement.

  11. Electroporation of DNA into Physarum polycephalum Mitochondria: Effects on Transcription and RNA Editing in Isolated Organelles

    Directory of Open Access Journals (Sweden)

    Jonatha M. Gott

    2016-12-01

    Full Text Available Mitochondrial RNAs in the acellular slime mold Physarum polycephalum contain nucleotides that are not encoded in the mitochondrial genes from which they are transcribed. These site-specific changes are quite extensive, comprising ~4% of the residues within mRNAs and ~2% of rRNAs and tRNAs. These “extra” nucleotides are added co-transcriptionally, but the means by which this is accomplished have not been elucidated. The cox1 mRNA also contains four sites of C to U changes, which occur post-transcriptionally, most likely via targeted deamination. The currently available in vitro systems for studying P. polycephalum editing are limited in that the template is the entire ~63,000 bp mitochondrial genome. This presents a significant challenge when trying to define the signals that specify editing sites. In an attempt to overcome this issue, a method for introducing DNA into isolated P. polycephalum mitochondria via electroporation has been developed. Exogenous DNA is expressed, but the transcripts synthesized from these templates are not edited under the conditions tested. However, transcripts derived from the mitochondrial genome are accurately edited after electroporation, indicating that the editing machinery is still functional. These findings suggest that this method may ultimately provide a feasible approach to elucidating editing signals.

  12. In vivo non-thermal irreversible electroporation impact on rat liver galvanic apparent internal resistance

    Energy Technology Data Exchange (ETDEWEB)

    Golberg, A; Laufer, S [Center for Bioengineering in the Service of Humanity and Society, School of Computer Science and Engineering, Hebrew University of Jerusalem, Jerusalem 91904 (Israel); Rabinowitch, H D [Robert H Smith Faculty of Agriculture, Food and Environment, Robert H Smith Institute of Plant Science and Genetics in Agriculture, Hebrew University of Jerusalem, Rehovot 76 100 (Israel); Rubinsky, B, E-mail: Rabin@agri.huji.ac.il [Department of Mechanical Engineering, Graduate Program in Biophysics, University of California at Berkeley, Berkeley, CA 84720 (United States)

    2011-02-21

    Non-thermal irreversible electroporation (NTIRE) is a biophysical phenomenon which involves application of electric field pulses to cells or tissues, causing certain rearrangements in the membrane structure leading to cell death. The treated tissue ac impedance changes induced by electroporation were shown to be the indicators for NTIRE efficiency. In a previous study we characterized in vitro tissue galvanic apparent internal resistance (GAIR) changes due to NTIRE. Here we describe an in vivo study in which we monitored the GAIR changes of a rat liver treated by NTIRE. Electrical pulses were delivered through the same Zn/Cu electrodes by which GAIR was measured. GAIR was measured before and for 3 h after the treatment at 15 min intervals. The results were compared to the established ac bioimpedance measurement method. A decrease of 33% was measured immediately after the NTIRE treatment and a 40% decrease was measured after 3 h in GAIR values; in the same time 40% and 47% decrease respectively were measured by ac bioimpedance analyses. The temperature increase due to the NTIRE was only 0.5 deg. C. The results open the way for an inexpensive, self-powered in vivo real-time NTIRE effectiveness measurement.

  13. Introduction of Foreign Genes into Silkworm Eggs by Electroporation and Its Application in Transgenic Vector Test

    Institute of Scientific and Technical Information of China (English)

    Xiu-Yang GUO; Liang DONG; Sheng-Peng WANG; Ting-Qing GUO; Jian-Yang WANG; Chang-De LU

    2004-01-01

    Electroporation as a methodology to introduce foreign genes into silkworm eggs was systematically analyzed. The foreign gene in both the newly hatched and 3rd instar larva DNA can be detected by PCR. The amount of foreign gene in 3rd instar larva DNA was about 1/1000 of that in newly hatched larva DNA. The ratio of foreign gene entering into silkworm eggs was voltage dependent and showed significant difference between the tested silkworm strains. When the piggyBac transposon system was applied, the effect of nuclear localization signal (NLS) peptide and the in vitro transcribed transposase mRNA on the transposition rate has been measured. Results showed that the in vitro transcribed transposase mRNA facilitated trans-position to take place earlier and NLS could result in higher transposition probability and earlier transposition as well. When linearized vectors containing varied length of flanking homologous sequences around a reporter gene were introduced into silkworm eggs by electroporation, the one with 2.6 kb total arm length gave higher G1 positive ratio than that with total arm length of 1.5 kb and 800 bp.

  14. Electroporation of DNA into Physarum polycephalum Mitochondria: Effects on Transcription and RNA Editing in Isolated Organelles.

    Science.gov (United States)

    Gott, Jonatha M; Naegele, Gregory M; Howell, Scott J

    2016-12-14

    Mitochondrial RNAs in the acellular slime mold Physarum polycephalum contain nucleotides that are not encoded in the mitochondrial genes from which they are transcribed. These site-specific changes are quite extensive, comprising ~4% of the residues within mRNAs and ~2% of rRNAs and tRNAs. These "extra" nucleotides are added co-transcriptionally, but the means by which this is accomplished have not been elucidated. The cox1 mRNA also contains four sites of C to U changes, which occur post-transcriptionally, most likely via targeted deamination. The currently available in vitro systems for studying P. polycephalum editing are limited in that the template is the entire ~63,000 bp mitochondrial genome. This presents a significant challenge when trying to define the signals that specify editing sites. In an attempt to overcome this issue, a method for introducing DNA into isolated P. polycephalum mitochondria via electroporation has been developed. Exogenous DNA is expressed, but the transcripts synthesized from these templates are not edited under the conditions tested. However, transcripts derived from the mitochondrial genome are accurately edited after electroporation, indicating that the editing machinery is still functional. These findings suggest that this method may ultimately provide a feasible approach to elucidating editing signals.

  15. Introduction of foreign DNA into the water flea, Daphnia magna, by electroporation.

    Science.gov (United States)

    Kato, Yasuhiko; Kobayashi, Kaoru; Watanabe, Hajime; Iguchi, Taisen

    2010-03-01

    Daphnids inhabit a diverse array of aquatic environments and they are a good model for understanding response and adaptation to environmental changes and they have been used one of standard organisms in ecotoxicology. Recent progress of genomics changed the tools for analyzing responses of daphnids, because gene expression changes can be observed before the emergence of prominent adverse effect such as immobility of the organism. Thus understanding of biological changes from gene expression level can be one of the sensitive tools for the evaluation of environmental response of organisms. However, there was no technique for genetic manipulation in daphnids. Hence, we have developed a gene introduction technique based on electroporation. There are two critical points for the successful introduction of foreign DNA into D. magna. (1) Injection of DNA into blood stream. (2) Usage of very low voltage for the electroporation. The injected DNA containing green fluorescent protein (GFP) could be introduced daphnids and the expression of GFP could be detected in living daphnids. This is the first report of gene introduction to daphnids and, together with the emerging genome sequences, will be useful for the expanding our use of daphnid in ecotoxicology.

  16. Genomic DNA extraction from cells by electroporation on an integrated microfluidic platform.

    Science.gov (United States)

    Geng, Tao; Bao, Ning; Sriranganathanw, Nammalwar; Li, Liwu; Lu, Chang

    2012-11-06

    The vast majority of genetic analysis of cells involves chemical lysis for release of DNA molecules. However, chemical reagents required in the lysis interfere with downstream molecular biology and often require removal after the step. Electrical lysis based on irreversible electroporation is a promising technique to prepare samples for genetic analysis due to its purely physical nature, fast speed, and simple operation. However, there has been no experimental confirmation on whether electrical lysis extracts genomic DNA from cells in a reproducible and efficient fashion in comparison to chemical lysis, especially for eukaryotic cells that have most of the DNA enclosed in the nucleus. In this work, we construct an integrated microfluidic chip that physically traps a low number of cells, lyses the cells using electrical pulses rapidly, then purifies and concentrates genomic DNA. We demonstrate that electrical lysis offers high efficiency for DNA extraction from both eukaryotic cells (up to ∼36% for Chinese hamster ovary cells) and bacterial cells (up to ∼45% for Salmonella typhimurium) that is comparable to the widely used chemical lysis. The DNA extraction efficiency has dependence on both the electric parameters and relative amount of beads used for DNA adsorption. We envision that electroporation-based DNA extraction will find use in ultrasensitive assays that benefit from minimal dilution and simple procedures.

  17. Regeneration of transformed shoots from electroporated soybean (Glycine max (L.) Merr.) protoplasts.

    Science.gov (United States)

    Dhir, S K; Dhir, S; Sturtevant, A P; Widholm, J M

    1991-06-01

    Stable transformation of soybean (Glycine max (L.) Merr.) protoplasts isolated from immature cotyledons was achieved following electroporation with plasmid DNA carrying chimeric genes encoding ß-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transformed colonies were stringently selected by growing 15-day-old protoplast-derived cells in the presence of 40 μg/ml of hygromycin-B for 6 weeks. Over 93% of the resistant cells and colonies exhibited GUS activity, indicating that the two marker genes borne on a single plasmid were co-introduced and co-expressed at a very high freguency. This transformation procedure reproducibly yields transformants at frequencies of 2.9-6.8 × 10(-4) (based on the number of protoplasts electroporated) or 23.0% (based on the number of control microcalli formed) counted after 6 weeks of selection. After repeated subculturing on regeneration medium, shoots were induced from 8.0% of the transformed calli. Southern hybridization confirmed the presence of both the GUS and hygromycin genes in the transformed calli and shoots.

  18. In vivo non-thermal irreversible electroporation impact on rat liver galvanic apparent internal resistance.

    Science.gov (United States)

    Golberg, A; Laufer, S; Rabinowitch, H D; Rubinsky, B

    2011-02-21

    Non-thermal irreversible electroporation (NTIRE) is a biophysical phenomenon which involves application of electric field pulses to cells or tissues, causing certain rearrangements in the membrane structure leading to cell death. The treated tissue ac impedance changes induced by electroporation were shown to be the indicators for NTIRE efficiency. In a previous study we characterized in vitro tissue galvanic apparent internal resistance (GAIR) changes due to NTIRE. Here we describe an in vivo study in which we monitored the GAIR changes of a rat liver treated by NTIRE. Electrical pulses were delivered through the same Zn/Cu electrodes by which GAIR was measured. GAIR was measured before and for 3 h after the treatment at 15 min intervals. The results were compared to the established ac bioimpedance measurement method. A decrease of 33% was measured immediately after the NTIRE treatment and a 40% decrease was measured after 3 h in GAIR values; in the same time 40% and 47% decrease respectively were measured by ac bioimpedance analyses. The temperature increase due to the NTIRE was only 0.5 °C. The results open the way for an inexpensive, self-powered in vivo real-time NTIRE effectiveness measurement.

  19. Expression of HTLV-1 Genes in T-Cells Using RNA Electroporation.

    Science.gov (United States)

    Manicone, Mariangela; Rende, Francesca; Cavallari, Ilaria; Thoma-Kress, Andrea K; Ciminale, Vincenzo

    2017-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) infects about 20 million people world-wide. Around 5% of the infected individuals develop adult T-cell leukemia (ATL) or a neurological disease termed tropical spastic paraparesis (TSP) after a clinical latency of years to decades. Through the use of two promoters and alternative splicing HTLV-1 expresses at least 12 different proteins. HTLV-1 establishes a life-long persistent infection by inducing the clonal expansion of infected cells, a property largely ascribed to the viral genes Tax and HBZ. However, the fact that ATL arises in a minority of infected individuals after a long clinical latency suggests the existence of factors counterbalancing the oncogenic potential of HTLV-1 in the context of natural infection.To study the role of the different HTLV-1 gene products in the HTLV-1 life cycle, we optimized a transfection protocol for primary T-cells using an approach based on the electroporation of in vitro-transcribed RNA. Results showed that the RNA transfection technique combines a high transfection efficiency with low toxicity, not only in Jurkat T-cells but also in primary T-cells. These findings suggest that RNA electroporation is preferable for experiments aimed at investigating the role of HTLV-1 gene products in the context of primary T-cells, which represent the main target of HTLV-1 in vivo.

  20. Single-cell detection of mRNA expression using nanofountain-probe electroporated molecular beacons.

    Science.gov (United States)

    Giraldo-Vela, Juan P; Kang, Wonmo; McNaughton, Rebecca L; Zhang, Xuemei; Wile, Brian M; Tsourkas, Andrew; Bao, Gang; Espinosa, Horacio D

    2015-05-01

    New techniques for single-cell analysis enable new discoveries in gene expression and systems biology. Time-dependent measurements on individual cells are necessary, yet the common single-cell analysis techniques used today require lysing the cell, suspending the cell, or long incubation times for transfection, thereby interfering with the ability to track an individual cell over time. Here a method for detecting mRNA expression in live single cells using molecular beacons that are transfected into single cells by means of nanofountain probe electroporation (NFP-E) is presented. Molecular beacons are oligonucleotides that emit fluorescence upon binding to an mRNA target, rendering them useful for spatial and temporal studies of live cells. The NFP-E is used to transfect a DNA-based beacon that detects glyceraldehyde 3-phosphate dehydrogenase and an RNA-based beacon that detects a sequence cloned in the green fluorescence protein mRNA. It is shown that imaging analysis of transfection and mRNA detection can be performed within seconds after electroporation and without disturbing adhered cells. In addition, it is shown that time-dependent detection of mRNA expression is feasible by transfecting the same single cell at different time points. This technique will be particularly useful for studies of cell differentiation, where several measurements of mRNA expression are required over time.

  1. Comparison between direct and reverse electroporation of cells in situ: a simulation study.

    Science.gov (United States)

    Towhidi, Leila; Khodadadi, Delaram; Maimari, Nataly; Pedrigi, Ryan M; Ip, Henry; Kis, Zoltan; Kwak, Brenda R; Petrova, Tatiana W; Delorenzi, Mauro; Krams, Rob

    2016-03-01

    The discovery of the human genome has unveiled new fields of genomics, transcriptomics, and proteomics, which has produced paradigm shifts on how to study disease mechanisms, wherein a current central focus is the understanding of how gene signatures and gene networks interact within cells. These gene function studies require manipulating genes either through activation or inhibition, which can be achieved by temporarily permeabilizing the cell membrane through transfection to delivercDNAorRNAi. An efficient transfection technique is electroporation, which applies an optimized electric pulse to permeabilize the cells of interest. When the molecules are applied on top of seeded cells, it is called "direct" transfection and when the nucleic acids are printed on the substrate and the cells are seeded on top of them, it is termed "reverse" transfection. Direct transfection has been successfully applied in previous studies, whereas reverse transfection has recently gained more attention in the context of high-throughput experiments. Despite the emerging importance, studies comparing the efficiency of the two methods are lacking. In this study, a model for electroporation of cells in situ is developed to address this deficiency. The results indicate that reverse transfection is less efficient than direct transfection. However, the model also predicts that by increasing the concentration of deliverable molecules by a factor of 2 or increasing the applied voltage by 20%, reverse transfection can be approximately as efficient as direct transfection.

  2. High-efficiency electroporation of the spinal cord in larval axolotl.

    Science.gov (United States)

    Rodrigo Albors, Aida; Tanaka, Elly M

    2015-01-01

    Axolotls are well known for their remarkable ability to regenerate complex body parts and structures throughout life, including the entire limb and tail. Particularly fascinating is their ability to regenerate a fully functional spinal cord after losing the tail. Electroporation of DNA plasmids or morpholinos is a valuable tool to gain mechanistic insight into the cellular and molecular basis of regeneration. It provides among other advantages a simple and fast method to test gene function in a temporally and spatially controlled manner. Some classic drawbacks of the method, such as low transfection efficiency and damage to the tissue, had hindered our understanding of the contribution of different signaling pathways to regeneration. Here, we describe a comprehensive protocol for electroporation of the axolotl spinal cord that overcomes this limitations using a combination of high-voltage and short-length pulses followed by lower-voltage and longer-length pulses. Our approach yields highly efficient transfection of spinal cord cells with minimal tissue damage, which now allows the molecular dissection of spinal cord regeneration.

  3. Optimising Antibiotic Usage to Treat Bacterial Infections

    Science.gov (United States)

    Paterson, Iona K.; Hoyle, Andy; Ochoa, Gabriela; Baker-Austin, Craig; Taylor, Nick G. H.

    2016-11-01

    The increase in antibiotic resistant bacteria poses a threat to the continued use of antibiotics to treat bacterial infections. The overuse and misuse of antibiotics has been identified as a significant driver in the emergence of resistance. Finding optimal treatment regimens is therefore critical in ensuring the prolonged effectiveness of these antibiotics. This study uses mathematical modelling to analyse the effect traditional treatment regimens have on the dynamics of a bacterial infection. Using a novel approach, a genetic algorithm, the study then identifies improved treatment regimens. Using a single antibiotic the genetic algorithm identifies regimens which minimise the amount of antibiotic used while maximising bacterial eradication. Although exact treatments are highly dependent on parameter values and initial bacterial load, a significant common trend is identified throughout the results. A treatment regimen consisting of a high initial dose followed by an extended tapering of doses is found to optimise the use of antibiotics. This consistently improves the success of eradicating infections, uses less antibiotic than traditional regimens and reduces the time to eradication. The use of genetic algorithms to optimise treatment regimens enables an extensive search of possible regimens, with previous regimens directing the search into regions of better performance.

  4. Ant Colony Optimisation for Backward Production Scheduling

    Directory of Open Access Journals (Sweden)

    Leandro Pereira dos Santos

    2012-01-01

    Full Text Available The main objective of a production scheduling system is to assign tasks (orders or jobs to resources and sequence them as efficiently and economically (optimised as possible. Achieving this goal is a difficult task in complex environment where capacity is usually limited. In these scenarios, finding an optimal solution—if possible—demands a large amount of computer time. For this reason, in many cases, a good solution that is quickly found is preferred. In such situations, the use of metaheuristics is an appropriate strategy. In these last two decades, some out-of-the-shelf systems have been developed using such techniques. This paper presents and analyses the development of a shop-floor scheduling system that uses ant colony optimisation (ACO in a backward scheduling problem in a manufacturing scenario with single-stage processing, parallel resources, and flexible routings. This scenario was found in a large food industry where the corresponding author worked as consultant for more than a year. This work demonstrates the applicability of this artificial intelligence technique. In fact, ACO proved to be as efficient as branch-and-bound, however, executing much faster.

  5. Noise aspects at aerodynamic blade optimisation projects

    Energy Technology Data Exchange (ETDEWEB)

    Schepers, J.G. [Netherlands Energy Research Foundation, Petten (Netherlands)

    1997-12-31

    This paper shows an example of an aerodynamic blade optimisation, using the program PVOPT. PVOPT calculates the optimal wind turbine blade geometry such that the maximum energy yield is obtained. Using the aerodynamic optimal blade design as a basis, the possibilities of noise reduction are investigated. The aerodynamic optimised geometry from PVOPT is the `real` optimum (up to the latest decimal). The most important conclusion from this study is, that it is worthwhile to investigate the behaviour of the objective function (in the present case the energy yield) around the optimum: If the optimum is flat, there is a possibility to apply modifications to the optimum configuration with only a limited loss in energy yield. It is obvious that the modified configurations emits a different (and possibly lower) noise level. In the BLADOPT program (the successor of PVOPT) it will be possible to quantify the noise level and hence to assess the reduced noise emission more thoroughly. At present the most promising approaches for noise reduction are believed to be a reduction of the rotor speed (if at all possible), and a reduction of the tip angle by means of low lift profiles, or decreased twist at the outboard stations. These modifications were possible without a significant loss in energy yield. (LN)

  6. CT-guided Irreversible Electroporation in an Acute Porcine Liver Model: Effect of Previous Transarterial Iodized Oil Tissue Marking on Technical Parameters, 3D Computed Tomographic Rendering of the Electroporation Zone, and Histopathology

    Energy Technology Data Exchange (ETDEWEB)

    Sommer, C. M., E-mail: christof.sommer@med.uni-heidelberg.de [University Hospital Heidelberg, Department of Diagnostic and Interventional Radiology (Germany); Fritz, S., E-mail: stefan.fritz@med.uni-heidelberg.de [University Hospital Heidelberg, Department of General Visceral and Transplantation Surgery (Germany); Vollherbst, D., E-mail: dominikvollherbst@web.de [University Hospital Heidelberg, Department of Diagnostic and Interventional Radiology (Germany); Zelzer, S., E-mail: s.zelzer@dkfz-heidelberg.de [German Cancer Research Center (dkfz), Medical and Biological Informatics (Germany); Wachter, M. F., E-mail: fredericwachter@googlemail.com; Bellemann, N., E-mail: nadine.bellemann@med.uni-heidelberg.de; Gockner, T., E-mail: theresa.gockner@med.uni-heidelberg.de; Mokry, T., E-mail: theresa.mokry@med.uni-heidelberg.de; Schmitz, A., E-mail: anne.schmitz@med.uni-heidelberg.de [University Hospital Heidelberg, Department of Diagnostic and Interventional Radiology (Germany); Aulmann, S., E-mail: sebastian.aulmann@mail.com [University Hospital Heidelberg, Department of General Pathology (Germany); Stampfl, U., E-mail: ulrike.stampfl@med.uni-heidelberg.de [University Hospital Heidelberg, Department of Diagnostic and Interventional Radiology (Germany); Pereira, P., E-mail: philippe.pereira@slk-kliniken.de [SLK Kliniken Heilbronn GmbH, Clinic for Radiology, Minimally-invasive Therapies and Nuclear Medicine (Germany); Kauczor, H. U., E-mail: hu.kauczor@med.uni-heidelberg.de [University Hospital Heidelberg, Department of Diagnostic and Interventional Radiology (Germany); Werner, J., E-mail: jens.werner@med.uni-heidelberg.de [University Hospital Heidelberg, Department of General Visceral and Transplantation Surgery (Germany); Radeleff, B. A., E-mail: boris.radeleff@med.uni-heidelberg.de [University Hospital Heidelberg, Department of Diagnostic and Interventional Radiology (Germany)

    2015-02-15

    PurposeTo evaluate the effect of previous transarterial iodized oil tissue marking (ITM) on technical parameters, three-dimensional (3D) computed tomographic (CT) rendering of the electroporation zone, and histopathology after CT-guided irreversible electroporation (IRE) in an acute porcine liver model as a potential strategy to improve IRE performance.MethodsAfter Ethics Committee approval was obtained, in five landrace pigs, two IREs of the right and left liver (RL and LL) were performed under CT guidance with identical electroporation parameters. Before IRE, transarterial marking of the LL was performed with iodized oil. Nonenhanced and contrast-enhanced CT examinations followed. One hour after IRE, animals were killed and livers collected. Mean resulting voltage and amperage during IRE were assessed. For 3D CT rendering of the electroporation zone, parameters for size and shape were analyzed. Quantitative data were compared by the Mann–Whitney test. Histopathological differences were assessed.ResultsMean resulting voltage and amperage were 2,545.3 ± 66.0 V and 26.1 ± 1.8 A for RL, and 2,537.3 ± 69.0 V and 27.7 ± 1.8 A for LL without significant differences. Short axis, volume, and sphericity index were 16.5 ± 4.4 mm, 8.6 ± 3.2 cm{sup 3}, and 1.7 ± 0.3 for RL, and 18.2 ± 3.4 mm, 9.8 ± 3.8 cm{sup 3}, and 1.7 ± 0.3 for LL without significant differences. For RL and LL, the electroporation zone consisted of severely widened hepatic sinusoids containing erythrocytes and showed homogeneous apoptosis. For LL, iodized oil could be detected in the center and at the rim of the electroporation zone.ConclusionThere is no adverse effect of previous ITM on technical parameters, 3D CT rendering of the electroporation zone, and histopathology after CT-guided IRE of the liver.

  7. Predicting electroporation of cells in an inhomogeneous electric field based on mathematical modeling and experimental CHO-cell permeabilization to propidium iodide determination.

    Science.gov (United States)

    Dermol, Janja; Miklavčič, Damijan

    2014-12-01

    High voltage electric pulses cause electroporation of the cell membrane. Consequently, flow of the molecules across the membrane increases. In our study we investigated possibility to predict the percentage of the electroporated cells in an inhomogeneous electric field on the basis of the experimental results obtained when cells were exposed to a homogeneous electric field. We compared and evaluated different mathematical models previously suggested by other authors for interpolation of the results (symmetric sigmoid, asymmetric sigmoid, hyperbolic tangent and Gompertz curve). We investigated the density of the cells and observed that it has the most significant effect on the electroporation of the cells while all four of the mathematical models yielded similar results. We were able to predict electroporation of cells exposed to an inhomogeneous electric field based on mathematical modeling and using mathematical formulations of electroporation probability obtained experimentally using exposure to the homogeneous field of the same density of cells. Models describing cell electroporation probability can be useful for development and presentation of treatment planning for electrochemotherapy and non-thermal irreversible electroporation.

  8. Multi-objective evolutionary optimisation for product design and manufacturing

    CERN Document Server

    2011-01-01

    Presents state-of-the-art research in the area of multi-objective evolutionary optimisation for integrated product design and manufacturing Provides a comprehensive review of the literature Gives in-depth descriptions of recently developed innovative and novel methodologies, algorithms and systems in the area of modelling, simulation and optimisation

  9. Optimisation of GnRH antagonist use in ART

    NARCIS (Netherlands)

    Hamdine, O.

    2014-01-01

    This thesis focuses on the optimisation of controlled ovarian stimulation for IVF using exogenous FSH and GnRH antagonist co-treatment, by studying the timing of the initiation of GnRH antagonist co-medication and the role of ovarian reserve markers in optimising ovarian response and reproductive ou

  10. Aerodynamic shape parameterisation and optimisation of novel configurations

    NARCIS (Netherlands)

    Straathof, M.H.; Van Tooren, M.J.L.; Voskuijl, M.; Koren, B.

    2008-01-01

    The Multi-Disciplinary Design Optimisation (MDO) process can be supported by partial automation of analysis and optimisation steps. Design and Engineering Engines (DEE) are useful concepts to structure this type of automation. Within the DEE, a product can be parameterically defined using Knowledge

  11. DACIA LOGAN LIVE AXLE OPTIMISATION USING COMPUTER GRAPHICS

    Directory of Open Access Journals (Sweden)

    KIRALY Andrei

    2017-05-01

    Full Text Available The paper presents some contributions to the calculus and optimisation of a live axle used at Dacia Logan using computer graphics software for creating the model and afterwards using FEA evaluation to determine the effectiveness of the optimisation. Thus using specialized computer software, a simulation is made and the results were compared to the measured real prototype.

  12. There, and Back Again Quantum Theory and Global Optimisation

    CERN Document Server

    Audenaert, K M R

    2004-01-01

    We consider a problem in quantum theory that can be formulated as an optimisation problem and present a global optimisation algorithm for solving it, the foundation of which relies in turn on a theorem from quantum theory. To wit, we consider the maximal output purity $\

  13. Kriging based robust optimisation algorithm for minimax problems in electromagnetics

    Directory of Open Access Journals (Sweden)

    Li Yinjiang

    2016-12-01

    Full Text Available The paper discusses some of the recent advances in kriging based worst-case design optimisation and proposes a new two-stage approach to solve practical problems. The efficiency of the infill points allocation is improved significantly by adding an extra layer of optimisation enhanced by a validation process.

  14. GAOS: Spatial optimisation of crop and nature within agricultural fields

    NARCIS (Netherlands)

    Bruin, de S.; Janssen, H.; Klompe, A.; Lerink, P.; Vanmeulebrouk, B.

    2010-01-01

    This paper proposes and demonstrates a spatial optimiser that allocates areas of inefficient machine manoeuvring to field margins thus improving the use of available space and supporting map-based Controlled Traffic Farming. A prototype web service (GAOS) allows farmers to optimise tracks within the

  15. Parameter Optimisation for the Behaviour of Elastic Models over Time

    DEFF Research Database (Denmark)

    Mosegaard, Jesper

    2004-01-01

    Optimisation of parameters for elastic models is essential for comparison or finding equivalent behaviour of elastic models when parameters cannot simply be transferred or converted. This is the case with a large range of commonly used elastic models. In this paper we present a general method...... that will optimise parameters based on the behaviour of the elastic models over time....

  16. Multi-criterion scantling optimisation of cruise ships

    OpenAIRE

    2010-01-01

    A numerical tool for the optimisation of the scantlings of a ship is extended by considering production cost, weight and moment of iner tia in the objective function. A multi-criteria optimisation of a passenger ship is conducted to illustrate the analysis process. Pareto frontiers are obtained and results are verified with Bureau Veritas rules.

  17. The Effect of Electroporation of a Lyotroic Liquid Crystal Genistein-Based Formulation in the Recovery of Murine Melanoma Lesions

    Directory of Open Access Journals (Sweden)

    Corina Danciu

    2015-07-01

    Full Text Available A lamellar lyotropic liquid crystal genistein-based formulation (LLC-Gen was prepared in order to increase the aqueous solubility of the lipophilic phytocompound genistein. The formulation was applied locally, in a murine model of melanoma, with or without electroporation. The results demonstrated that, when the formulation was applied by electroporation, the tumors appeared later. During the 21 days of the experiment, the LLC-Gen formulation decreased the tumor volume, the amount of melanin and the degree of erythema, but when electroporation was applied, all these parameters indicated a better prognosis even (lower tumor volume, amount of melanin and degree of erythema. Although hematoxylin–eosin (HE staining confirmed the above events, application of the LLC-Gen formulation by electroporation did not lead to a significant effect in terms of the serum concentrations of the protein S100B and serum neuron specific enolase (NSE, or the tissue expression of the platelet-derived growth factor receptor β (PDGFRβ antibody.

  18. An improved protocol for generation of immuno-potent dendritic cells through direct electroporation of CD14+monocytes

    NARCIS (Netherlands)

    Milano, Francesca; van Baal, Jantine W. P. M.; Rygiel, Agnieszka M.; Bergman, Jacques J. G. H. M.; Van Deventer, Sander J. H.; Kapsenberg, Martien L.; Peppelenbosch, Maikel P.; Krishnadath, Kausilia K.

    2007-01-01

    In this study we demonstrate a novel protocol showing that electroporation of CD14+ monocytes directly isolated from blood with green fluorescent protein (GFP) RNA results in a 3-fold higher yield of antigen presenting dendritic cells (DCs) when compared to conventional methods employing immature DC

  19. Studies on mRNA electroporation of immature and mature dendritic cells: Effects on their immunogenic potential

    DEFF Research Database (Denmark)

    Met, O.; Eriksen, J.; Svane, Inge Marie

    2008-01-01

    Previous studies have shown that mRNA-electroporated dendritic cells (DCs) are able to process and present tumor-associated antigens, leading to the activation of tumor-specific T cells in vitro and in vivo. However, the optimal maturation state of antigen loading and half-life of the m...

  20. Thermal damage reduction associated with in vivo skin electroporation: A numerical investigation justifying aggressive pre-cooling

    Energy Technology Data Exchange (ETDEWEB)

    Becker, S.M.; Kuznetsov, A.V. [North Carolina State University, Raleigh (United States). Mechanical and Aerospace Engineering

    2007-01-15

    Electroporation is an approach used to enhance transdermal transport of large molecules in which the skin is exposed to a series of electric pulses. Electroporation temporarily destabilizes the structure of the outer skin layer, the stratum corneum, by creating microscopic pores through which agents, which ordinarily are unable to pass into the skin, are able to pass through this outer barrier. Of possible concern when exposing biological tissue to an electric field is thermal tissue damage associated with Joule heating. In order to find the electrical and transient thermal solutions associated with this process, this study develops a three-dimensional transient finite-volume composite model of in vivo skin electroporation. The electroporation process modeled consists of five 150ms long DC square wave pulses administered at 1-s intervals with an applied voltage of 400V. This paper finds that minor thermal influence of the electrode plate and the of a small presence blood vessel have a large impact on thermal damage. An aggressive pre-cooling technique is presented which is shown to dramatically reduce the risk of thermal damage. (author)

  1. Electroporation-based delivery of cell-penetrating peptide conjugates of peptide nucleic acids for antisense inhibition of intracellular bacteria.

    Science.gov (United States)

    Ma, Sai; Schroeder, Betsy; Sun, Chen; Loufakis, Despina Nelie; Cao, Zhenning; Sriranganathan, Nammalwar; Lu, Chang

    2014-10-01

    Cell penetrating peptides (CPPs) have been used for a myriad of cellular delivery applications and were recently explored for delivery of antisense agents such as peptide nucleic acids (PNAs) for bacterial inhibition. Although these molecular systems (i.e. CPP-PNAs) have shown ability to inhibit growth of bacterial cultures in vitro, they show limited effectiveness in killing encapsulated intracellular bacteria in mammalian cells such as macrophages, presumably due to difficulty involved in the endosomal escape of the reagents. In this report, we show that electroporation delivery dramatically increases the bioavailability of CPP-PNAs to kill Salmonella enterica serovar Typhimurium LT2 inside macrophages. Electroporation delivers the molecules without involving endocytosis and greatly increases the antisense effect. The decrease in the average number of Salmonella per macrophage under a 1200 V cm(-1) and 5 ms pulse was a factor of 9 higher than that without electroporation (in an experiment with a multiplicity of infection of 2 : 1). Our results suggest that electroporation is an effective approach for a wide range of applications involving CPP-based delivery. The microfluidic format will allow convenient functional screening and testing of PNA-based reagents for antisense applications.

  2. The site of administration influences both the type and the magnitude of the immune response induced by DNA vaccine electroporation.

    Science.gov (United States)

    Vandermeulen, Gaëlle; Vanvarenberg, Kevin; De Beuckelaer, Ans; De Koker, Stefaan; Lambricht, Laure; Uyttenhove, Catherine; Reschner, Anca; Vanderplasschen, Alain; Grooten, Johan; Préat, Véronique

    2015-06-22

    We investigated the influence of the site of administration of DNA vaccine on the induced immune response. DNA vaccines were administered by electroporation at three different sites: tibial cranial muscle, abdominal skin and ear pinna. Aiming to draw general conclusions about DNA vaccine delivery, we successively used several plasmids encoding either luciferase and ovalbumin as models or gp160 and P1A as vaccines against HIV and P815 mastocytoma, respectively. Low levels and duration of luciferase transgene expression were observed after electroporation of the abdominal skin, partly explaining its lower immunogenic performance as compared to the other sites of administration. Analyses of OT-I CD8+ and OT-II CD4+ T cell responses highlighted the differential impact of the delivery site on the elicited immune response. Muscle electroporation induced the strongest humoral immune response and both muscle and ear pinna sites induced cellular immunity against gp160. Ear pinna delivery generated the highest level of CTL responses against P1A but electroporation of muscle and ear pinna were equally efficient in delaying P815 growth and improving mice survival. The present study demonstrated that the site of administration is a key factor to be tested in the development of DNA vaccine.

  3. Efficient large volume electroporation of dendritic cells through micrometer scale manipulation of flow in a disposable polymer chip

    DEFF Research Database (Denmark)

    Selmeczi, David; Hansen, Thomas; Met, Özcan

    2011-01-01

    We present a hybrid chip of polymer and stainless steel designed for high-throughput continuous electroporation of cells in suspension. The chip is constructed with two parallel stainless steel mesh electrodes oriented perpendicular to the liquid flow. The relatively high hydrodynamic resistance ...

  4. Ca2+ uptake and cellular integrity in rat EDL muscle exposed to electrostimulation, electroporation, or A23187

    DEFF Research Database (Denmark)

    Gissel, Hanne; Clausen, Torben

    2003-01-01

    We tested the hypothesis that increased Ca2+ uptake in rat extensor digitorum longus (EDL) muscle elicits cell membrane damage as assessed from release of the intracellular enzyme lactate dehydrogenase (LDH). This was done by using 1) electrostimulation, 2) electroporation, and 3) the Ca2...

  5. Modified cuckoo search: A new gradient free optimisation algorithm

    Energy Technology Data Exchange (ETDEWEB)

    Walton, S., E-mail: 512465@swansea.ac.uk [College of Engineering, Swansea University, Swansea SA2 8PP, Wales (United Kingdom); Hassan, O.; Morgan, K.; Brown, M.R. [College of Engineering, Swansea University, Swansea SA2 8PP, Wales (United Kingdom)

    2011-09-15

    Highlights: > Modified cuckoo search (MCS) is a new gradient free optimisation algorithm. > MCS shows a high convergence rate, able to outperform other optimisers. > MCS is particularly strong at high dimension objective functions. > MCS performs well when applied to engineering problems. - Abstract: A new robust optimisation algorithm, which can be regarded as a modification of the recently developed cuckoo search, is presented. The modification involves the addition of information exchange between the top eggs, or the best solutions. Standard optimisation benchmarking functions are used to test the effects of these modifications and it is demonstrated that, in most cases, the modified cuckoo search performs as well as, or better than, the standard cuckoo search, a particle swarm optimiser, and a differential evolution strategy. In particular the modified cuckoo search shows a high convergence rate to the true global minimum even at high numbers of dimensions.

  6. Optimisation of a Crossdocking Distribution Centre Simulation Model

    CERN Document Server

    Adewunmi, Adrian

    2010-01-01

    This paper reports on continuing research into the modelling of an order picking process within a Crossdocking distribution centre using Simulation Optimisation. The aim of this project is to optimise a discrete event simulation model and to understand factors that affect finding its optimal performance. Our initial investigation revealed that the precision of the selected simulation output performance measure and the number of replications required for the evaluation of the optimisation objective function through simulation influences the ability of the optimisation technique. We experimented with Common Random Numbers, in order to improve the precision of our simulation output performance measure, and intended to use the number of replications utilised for this purpose as the initial number of replications for the optimisation of our Crossdocking distribution centre simulation model. Our results demonstrate that we can improve the precision of our selected simulation output performance measure value using C...

  7. Open Cut resource Optimisation as applied to coal

    Institute of Scientific and Technical Information of China (English)

    Martin L.Smith

    2007-01-01

    Pit optimisation is the earliest and most established application of its kind in the minerals industry,but this has been primarily driven by metal,not coal.Coal has the same financiaI drivers for resource optimisation as does the metalliferous industry,yet pit optimisation is not common practice.Why? The following discussion presents the basics of pit optimisation as they relate to coal and illustrates how a technology developed for massive deposits is not suitable for thin.multi-seam deposits where mine planning is often driven more by product quality than by value drivers such as Net Present Value.An alternative methodology is presented that takes advantage of the data structure of bedded deposits to optimise resource recovery in terms of a production schedule that meets constraints on coal quality.

  8. Numerical modeling of bi-polar (AC) pulse electroporation of single cell in microchannel to create nanopores on its membrane.

    Science.gov (United States)

    Movahed, Saeid; Bazargan-Lari, Yousef; Daneshmad, Farhang; Mashhoodi, Mashhood

    2014-12-01

    AC electroporation of a single cell in a microchannel was numerically studied. A 15 μm diameter cell was considered in a microchannel 25 μm in height and the influences of AC electric pulse on its membrane were numerically investigated. The cell was assumed to be suspended between two electroporative electrodes embedded on the walls of a microchannel. An amplitude and a time span of applied electric pulse were chosen to be 80 kV/m and 10 μs, respectively. For different frequency values (50, 100, 200, and 500 kHz), simulations were performed to show how the cell membrane was electroporated and the creation of nanopores. Obtained numerical results show that the most and the largest nanopores are created around poles of cell (nearest points of cell membrane to the electrodes). The numerical simulations also demonstrate that increased frequency will slightly decrease electroporated area of the cell membrane; additionally, growth of the created nanopores will be stabilized. It has also been proven that size and number of the created nanopores will be decreased by moving from the poles to the equator of the cell. There is almost no nanopore created in the vicinity of the equator. Frequency affects the rate of generation of nanopores. In case of AC electroporation, creation of nanopores has two phases that periodically repeat over time. In each period, the pore density sharply increases and then becomes constant. Enhancement of the frequency will result in decrease in time span of the periods. In each period, size of the created nanopores sharply increases and then slightly decreases. However, until the AC electric pulse is present, overall trends of creation and development of nanopores will be ascending. Variation of the size and number of created nanopores can be explained by considering time variation of transmembrane potential (difference of electric potential on two sides of cell membrane) which is clear in the results presented in this study.

  9. ATLAS software configuration and build tool optimisation

    CERN Document Server

    Rybkin, G; The ATLAS collaboration

    2014-01-01

    ATLAS software code base is over 6 million lines organised in about 2000 packages. It makes use of some 100 external software packages, is developed by more than 400 developers and used by more than 2500 physicists from over 200 universities and laboratories in 6 continents. To meet the challenge of configuration and building of this software, the Configuration Management Tool (CMT) is used. CMT expects each package to describe its build targets, build and environment setup parameters, dependencies on other packages in a text file called requirements, and each project (group of packages) to describe its policies and dependencies on other projects in a text project file. Based on the effective set of configuration parameters read from the requirements files of dependent packages and project files, CMT commands build the packages, generate the environment for their use, or query the packages. The main focus was on build time performance that was optimised within several approaches: reduction of the number of re...

  10. ATLAS software configuration and build tool optimisation

    CERN Document Server

    Rybkin, G; The ATLAS collaboration

    2013-01-01

    ATLAS software code base is over 6 million lines organised in about 2000 packages. It makes use of some 100 external software packages, is developed by more than 400 developers and used by more than 2500 physicists from over 200 universities and laboratories in 6 continents. To meet the challenge of configuration and building of this software, the Configuration Management Tool (CMT) is used. CMT expects each package to describe its build targets, build and environment setup parameters, dependencies on other packages in a text file called requirements, and each project (group of packages) to describe its policies and dependencies on other projects in a text project file. Based on the effective set of configuration parameters read from the requirements files of dependent packages and project files, CMT commands build the packages, generate the environment for their use, or query the packages. The main focus was on build time performance that was optimised within several approaches: reduction of the number of re...

  11. Specification, Verification and Optimisation of Business Processes

    DEFF Research Database (Denmark)

    Herbert, Luke Thomas

    to model checking. This allows for a rich set of both qualitative and quantitative properties of a business process to be precisely determined in an automated fashion directly from the model of the business process. A number of advanced applications of this framework are presented which allow for automated...... Model and Notation (BPMN). The automated analysis of business processes is done by means of quantitative probabilistic model checking which allows verification of validation and performance properties through use of an algorithm for the translation of business process models into a format amenable......This thesis develops a unified framework wherein to specify, verify and optimise stochastic business processes. This framework provides for the modelling of business processes via a mathematical structure which captures business processes as a series of connected activities. This structure...

  12. FEM Optimisation of Spark Plasma Sintering Furnace

    CERN Document Server

    Kellari, Demetrios Vasili

    2013-01-01

    Coupled electro-thermal FEM analysis has been carried out on a sintering furnace used to produce new materials for LHC collimators. The analysis showed there exist margins for improvement of the current process and equipment through minor changes. To optimise the design of the furnace several design changes have been proposed including: optimization of material selection using copper cooling plates, control of convection in cooling plates by lowering the water flow rate, modifying the electrode shape using unsymmetrical electrodes and upgrading the thermal shielding to make use of multilayer graphite shields. The results show that we have a significant improvement in temperature gradient on the plate, from 453 to 258 °C and a reduction in power requirement from 62 to 44 kW.

  13. Niobium Cavity Electropolishing Modelling and Optimisation

    CERN Document Server

    Ferreira, L M A; Forel, S; Shirra, J A

    2013-01-01

    It’s widely accepted that electropolishing (EP) is the most suitable surface finishing process to achieve high performance bulk Nb accelerating cavities. At CERN and in preparation for the processing of the 704 MHz high-beta Superconducting Proton Linac (SPL) cavities a new vertical electropolishing facility has been assembled and a study is on-going for the modelling of electropolishing on cavities with COMSOL® software. In a first phase, the electrochemical parameters were taken into account for a fixed process temperature and flow rate, and are presented in this poster as well as the results obtained on a real SPL single cell cavity. The procedure to acquire the data used as input for the simulation is presented. The modelling procedure adopted to optimise the cathode geometry, aimed at a uniform current density distribution in the cavity cell for the minimum working potential and total current is explained. Some preliminary results on fluid dynamics is also briefly described.

  14. Improving and optimising road pricing in Copenhagen

    DEFF Research Database (Denmark)

    Nielsen, Otto Anker; Larsen, Marie Karen

    2008-01-01

    though quite a number of proposed charging systems have been examined only a few pricing strategies have been investigated. This paper deals with the optimisation of different designs for a road pricing system in the Greater Copenhagen area with respect to temporal and spatial differentiation......The question whether to introduce toll rings or road pricing in Copenhagen has been discussed intensively during the last 10 years. The main results of previous analyses are that none of the systems would make a positive contribution at present, when considered from a socio-economic view. Even...... of the pricing levels. A detailed transport model was used to describe the demand effects. The model was based on data from a real test of road pricing on 500 car drivers. The paper compares the price systems with regard to traffic effects and generalised costs for users and society. It is shown how important...

  15. A code for optimising triplet layout

    CERN Document Server

    Van Riesen-Haupt, Leon; Abelleira, Jose; Cruz Alaniz, Emilia

    2017-01-01

    One of the main challenges when designing final focus systems of particle accelerators is maximising the beam stay clear in the strong quadrupole magnets of the inner triplet. Moreover it is desirable to keep the quadrupoles in the inner triplet as short as possible for space and costs reasons but also to reduce chromaticity and simplify corrections schemes. An algorithm that explores the triplet parameter space to optimise both these aspects was written. It uses thin lenses as a first approximation for a broad parameter scan and MADX for more precise calculations. The thin lens algorithm is significantly faster than a full scan using MADX and relatively precise at indicating the approximate area where the optimum solution lies.

  16. Optimising Signalised Intersection Using Wireless Vehicle Detectors

    DEFF Research Database (Denmark)

    Adjin, Daniel Michael Okwabi; Torkudzor, Moses; Asare, Jack

    Traffic congestion on roads wastes travel times. In this paper, we developed a vehicular traffic model to optimise a signalised intersection in Accra, using wireless vehicle detectors. Traffic volume gathered was extrapolated to cover 2011 and 2016 and were analysed to obtain the peak hour traffic...... volume causing congestion. The intersection was modelled and simulated in Synchro7 as an actuated signalised model using results from the analysed data. The model for morning peak periods gave optimal cycle lengths of 100s and 150s with corresponding intersection delay of 48.9s and 90.6s in 2011 and 2016...... respectively while that for the evening was 55s giving delay of 14.2s and 16.3s respectively. It is shown that the model will improve traffic flow at the intersection....

  17. Simulation and optimisation of turbulence in stellarators

    Energy Technology Data Exchange (ETDEWEB)

    Xanthopoulos, Pavlos; Helander, Per; Turkin, Yuriy; Plunk, Gabriel G.; Bird, Thomas; Proll, Josefine H.E. [Max-Planck-Institut fuer Plasmaphysik, EURATOM Association, Wendelsteinstr. 1, 17491 Greifswald (Germany); Mynick, Harry [Plasma Physics Laboratory, Princeton University, Princeton, New Jersey 08543 (United States); Jenko, Frank; Goerler, Tobias; Told, Daniel [Max-Planck-Institut fuer Plasmaphysik, EURATOM Association, Boltzmannstr. 2, 85748 Garching (Germany)

    2014-07-01

    In tokamaks and stellarators - two leading types of devices used in fusion research - magnetic field lines trace out toroidal surfaces on which the plasma density and temperature are constant, but turbulent fluctuations carry energy across these surfaces to the wall, thus degrading the plasma confinement. Using petaflop-scale simulations, we calculate for the first time the pattern of turbulent structures forming on stellarator magnetic surfaces, and find striking differences relative to tokamaks. The observed sensitivity of the turbulence to the magnetic geometry suggests that there is room for further confinement improvement, in addition to measures already taken to minimise the laminar transport. With an eye towards fully optimised stellarators, we present a proof-of-principle configuration with substantially reduced turbulence compared to an existing design.

  18. Optimisation of Multilayer Insulation an Engineering Approach

    CERN Document Server

    Chorowski, M; Parente, C; Riddone, G

    2001-01-01

    A mathematical model has been developed to describe the heat flux through multilayer insulation (MLI). The total heat flux between the layers is the result of three distinct heat transfer modes: radiation, residual gas conduction and solid spacer conduction. The model describes the MLI behaviour considering a layer-to-layer approach and is based on an electrical analogy, in which the three heat transfer modes are treated as parallel thermal impedances. The values of each of the transfer mode vary from layer to layer, although the total heat flux remains constant across the whole MLI blanket. The model enables the optimisation of the insulation with regard to different MLI parameters, such as residual gas pressure, number of layers and boundary temperatures. The model has been tested with experimental measurements carried out at CERN and the results revealed to be in a good agreement, especially for insulation vacuum between 10-5 Pa and 10-3 Pa.

  19. Common mode chokes and optimisation aspects

    Science.gov (United States)

    Kut, T.; Lücken, A.; Dickmann, S.; Schulz, D.

    2014-11-01

    Due to the increasing electrification of modern aircraft, as a result of the More Electric Aircraft concept, new strategies and approaches are required to fulfil the strict EMC aircraft standards (DO-160/ED-14-Sec. 20). Common mode chokes are a key component of electromagnetic filters and often oversized because of the unknown impedance of the surrounding power electronic system. This oversizing results in an increase of weight and volume. It has to be avoided as far as possible for mobile applications. In this context, an advanced method is presented to measure these impedances under operating conditions. Furthermore, the different parameters of the inductance design is explained and an optimisation for weight and volume is introduced.

  20. Analysis of retinal cell development in chick embryo by immunohistochemistry and in ovo electroporation techniques

    Directory of Open Access Journals (Sweden)

    Pashkova Anna

    2010-01-01

    Full Text Available Abstract Background Retinal cell development has been extensively investigated; however, the current knowledge of dynamic morphological and molecular changes is not yet complete. Results This study was aimed at revealing the dynamic morphological and molecular changes in retinal cell development during the embryonic stages using a new method of targeted retinal injection, in ovo electroporation, and immunohistochemistry techniques. A plasmid DNA that expresses the green fluorescent protein (GFP as a marker was delivered into the sub-retinal space to transfect the chick retinal stem/progenitor cells at embryonic day 3 (E3 or E4 with the aid of pulses of electric current. The transfected retinal tissues were analyzed at various stages during chick development from near the start of neurogenesis at E4 to near the end of neurogenesis at E18. The expression of GFP allowed for clear visualization of cell morphologies and retinal laminar locations for the indication of retinal cell identity. Immunohistochemistry using cell type-specific markers (e.g., Visinin, Xap-1, Lim1+2, Pkcα, NeuN, Pax6, Brn3a, Vimentin, etc. allowed further confirmation of retinal cell types. The composition of retinal cell types was then determined over time by counting the number of GFP-expressing cells observed with morphological characteristics specific to the various retinal cell types. Conclusion The new method of retinal injection and electroporation at E3 - E4 allows the visualization of all retinal cell types, including the late-born neurons, e.g., bipolar cells at a level of single cells, which has been difficult with a conventional method with injection and electroporation at E1.5. Based on data collected from analyses of cell morphology, laminar locations in the retina, immunohistochemistry, and cell counts of GFP-expressing cells, the time-line and dynamic morphological and molecular changes of retinal cell development were determined. These data provide more

  1. MRI study on reversible and irreversible electroporation induced blood brain barrier disruption.

    Directory of Open Access Journals (Sweden)

    Mohammad Hjouj

    Full Text Available Electroporation, is known to induce cell membrane permeabilization in the reversible (RE mode and cell death in the irreversible (IRE mode. Using an experimental system designed to produce a continuum of IRE followed by RE around a single electrode we used MRI to study the effects of electroporation on the brain. Fifty-four rats were injected with Gd-DOTA and treated with a G25 electrode implanted 5.5 mm deep into the striata. MRI was acquired immediately after treatment, 10 min, 20 min, 30 min, and up to three weeks following the treatment using: T1W, T2W, Gradient echo (GE, serial SPGR (DCE-MRI with flip angles ranging over 5-25°, and diffusion-weighted MRI (DWMRI. Blood brain barrier (BBB disruption was depicted as clear enhancement on T1W images. The average signal intensity in the regions of T1-enhancement, representing BBB disruption, increased from 1887±83 (arbitrary units immediately post treatment to 2246±94 20 min post treatment, then reached a plateau towards the 30 min scan where it reached 2289±87. DWMRI at 30 min showed no significant effects. Early treatment effects and late irreversible damage were clearly depicted on T2W. The enhancing volume on T2W has increased by an average of 2.27±0.27 in the first 24-48 hours post treatment, suggesting an inflammatory tissue response. The permanent tissue damage, depicted as an enhancing region on T2W, 3 weeks post treatment, decreased to an average of 50±10% of the T2W enhancing volumes on the day of the treatment which was 33±5% of the BBB disruption volume. Permanent tissue damage was significantly smaller than the volume of BBB disruption, suggesting, that BBB disruption is associated with RE while tissue damage with IRE. These results demonstrate the feasibility of applying reversible and irreversible electroporation for transient BBB disruption or permanent damage, respectively, and applying MRI for planning/monitoring disruption volume/shape by optimizing electrode positions

  2. A statistical model for multidimensional irreversible electroporation cell death in tissue

    Directory of Open Access Journals (Sweden)

    Rubinsky Boris

    2010-02-01

    Full Text Available Abstract Background Irreversible electroporation (IRE is a minimally invasive tissue ablation technique which utilizes electric pulses delivered by electrodes to a targeted area of tissue to produce high amplitude electric fields, thus inducing irreversible damage to the cell membrane lipid bilayer. An important application of this technique is for cancer tissue ablation. Mathematical modelling is considered important in IRE treatment planning. In the past, IRE mathematical modelling used a deterministic single value for the amplitude of the electric field required for causing cell death. However, tissue, particularly cancerous tissue, is comprised of a population of different cells of different sizes and orientations, which in conventional IRE are exposed to complex electric fields; therefore, using a deterministic single value is overly simplistic. Methods We introduce and describe a new methodology for evaluating IRE induced cell death in tissue. Our approach employs a statistical Peleg-Fermi model to correlate probability of cell death in heterogeneous tissue to the parameters of electroporation pulses such as the number of pulses, electric field amplitude and pulse length. For treatment planning, the Peleg-Fermi model is combined with a numerical solution of the multidimensional electric field equation cast in a dimensionless form. This is the first time in which this concept is used for evaluating IRE cell death in multidimensional situations. Results We illustrate the methodology using data reported in literature for prostate cancer cell death by IRE. We show how to fit this data to a Fermi function in order to calculate the critical statistic parameters. To illustrate the use of the methodology, we simulated 2-D irreversible electroporation protocols and produced 2-D maps of the statistical distribution of cell death in the treated region. These plots were compared to plots produced using a deterministic model of cell death by IRE and

  3. Genetic transformation of marine cyanobacterium Synechococcus sp. CC9311 (Cyanophyceae) by electroporation

    Science.gov (United States)

    Chen, Huaxin; Lin, Hanzhi; Jiang, Peng; Li, Fuchao; Qin, Song

    2013-03-01

    Synechococcus sp. CC9311 is a marine cyanobacterium characterized by type IV chromatic acclimation (CA). A genetic transformation system was developed as a first step to elucidate the molecular mechanism of CA. The results show that Synechococcus sp. CC9311 cells were sensitive to four commonly used antibiotics: ampicillin, kanamycin, spectinomycin, and chloramphenicol. An integrative plasmid to disrupt the putative phycoerythrin lyase gene mpeV, using a kanamycin resistance gene as selectable marker, was constructed by recombinant polymerase chain reaction. The plasmid was then transformed into Synechococcus sp. CC9311 via electroporation. High transformation efficiency was achieved at a field strength of 2 kV/cm. DNA analysis showed that mpeV was fully disrupted following challenge of the transformants with a high concentration of kanamycin. In addition, the transformants that displayed poor growth on agar SN medium could be successfully plated on agarose SN medium.

  4. DNA delivery into anterior neural tube of zebrafish embryos by electroporation.

    Science.gov (United States)

    Teh, Cathleen; Chong, Shang Wei; Korzh, Vladimir

    2003-11-01

    The zebrafish is widely used for functional studies of vertebrate genes. It is accessible to manipulations during all stages of embryogenesis because the embryo develops externally and is optically transparent. However, functional studies conducted on the zebrafish have been generally limited to the earliest phase of activity of the gene of interest, which is a limitation in studies of genes that are expressed at various stages of embryonic development. It is therefore necessary to develop methods that allow for the modulation of gene activity during later stages of zebrafish development while leaving earlier functions intact. We have successfully electroporated the green fluorescent protein (GFP) reporter gene into the neural tube of the zebrafish embryo in a unidirectional or bilateral manner. This approach can be used for the functional analysis of the late role of developmental genes in the neural tube of zebrafish embryo and larvae.

  5. DNA transfection of bone marrow mesenchymal stem cells using micro electroporation chips

    KAUST Repository

    Deng, Peigang

    2011-02-01

    Experimental study of electroporation of bone marrow mesenchymal stem cells (MSCs) at the single-cell level was carried out on a micro EP chip by using single electric rectangular pulse. The threshold values of the electrode potential and pulse width for gas bubble generation on the micro electrodes due to electrolysis of water were revealed as 4.5 volt and 100 μs, respectively. Quantitative EP study was performed with various electric field strengths for various pulse widths, ranging from 20μs to 15ms. Over 1,000 single-cell EP results were used to construct an EP "phase diagram", which delineates the boundaries for (1) effective EP of MSCs and (2) electric cell lysis of MSCs. Finally, the micro EP chip showed successful transfection of the pEGFP-C1 plasmid into the MSCs by properly choosing the electric parameters from the EP "phase diagram". © 2011 IEEE.

  6. Genetic transformation of marine cyanobacterium Synechococcus sp.CC9311 (Cyanophyceae) by electroporation

    Institute of Scientific and Technical Information of China (English)

    CHEN Huaxin; LIN Hanzhi; JIANG Peng; LI Fuchao; QIN Song

    2013-01-01

    Synechococcus sp.CC9311 is a marine cyanobacterium characterized by type V chromatic acclimation (CA).A genetic transformation system was developed as a first step to elucidate the molecular mechanism of CA.The results show that Synechococcus sp.CC9311 cells were sensitive to four commonly used antibiotics:ampicillin,kanamycin,spectinomycin,and chloramphenicol.An integrative plasmid to disrupt the putative phycoerythrin lyase gene mpeV,using a kanamycin resistance gene as selectable marker,was constructed by recombinant polymerase chain reaction.The plasmid was then transformed into Synechococcus sp.CC9311 via electroporation.High transformation efficiency was achieved at a field strength of 2 kV/cm.DNA analysis showed that rpe V was fully disrupted following challenge of the transformants with a high concentration of kanamycin.In addition,the transformants that displayed poor growth on agar SN medium could be successfully plated on agarose SN medium.

  7. Percutaneous Irreversible Electroporation of Locally Advanced Pancreatic Carcinoma Using the Dorsal Approach: A Case Report

    Energy Technology Data Exchange (ETDEWEB)

    Scheffer, Hester J., E-mail: hj.scheffer@vumc.nl; Melenhorst, Marleen C. A. M., E-mail: m.melenhorst@vumc.nl [VU University Medical Center, Department of Radiology and Nuclear Medicine (Netherlands); Vogel, Jantien A., E-mail: j.a.vogel@amc.uva.nl [Academic Medical Center, Department of Surgery (Netherlands); Tilborg, Aukje A. J. M. van, E-mail: a.vantilborg@vumc.nl [VU University Medical Center, Department of Radiology and Nuclear Medicine (Netherlands); Nielsen, Karin, E-mail: k.nielsen@vumc.nl; Kazemier, Geert, E-mail: g.kazemier@vumc.nl [VU University Medical Center, Department of Surgery (Netherlands); Meijerink, Martijn R., E-mail: mr.meijerink@vumc.nl [VU University Medical Center, Department of Radiology and Nuclear Medicine (Netherlands)

    2015-06-15

    Irreversible electroporation (IRE) is a novel image-guided ablation technique that is increasingly used to treat locally advanced pancreatic carcinoma (LAPC). We describe a 67-year-old male patient with a 5 cm stage III pancreatic tumor who was referred for IRE. Because the ventral approach for electrode placement was considered dangerous due to vicinity of the tumor to collateral vessels and duodenum, the dorsal approach was chosen. Under CT-guidance, six electrodes were advanced in the tumor, approaching paravertebrally alongside the aorta and inferior vena cava. Ablation was performed without complications. This case describes that when ventral electrode placement for pancreatic IRE is impaired, the dorsal approach could be considered alternatively.

  8. Effect of irreversible electroporation on three-dimensional cell culture model.

    Science.gov (United States)

    Kurata, Kosaku; Matsushita, Masahiro; Yoshii, Takashi; Fukunaga, Takanobu; Takamatsu, Hiroshi

    2012-01-01

    Irreversible electroporation (IRE) is a new treatment to necrotize abnormal cells by high electric pulses. Electric potential difference over 1 V across the plasma membrane permanently permeabilizes the cell with keeping the extracellular matrix intact if the thermal damage due to the Joule heating effect is avoided. This is the largest advantage of the IRE compared to the other conventional treatment. However, since the IRE has just started to be used in clinical tests, it is important to predict the necrotized region that depends on pulse parameters and electrode arrangement. We therefore examined the numerical solution to the Laplace equation for the static electric field to predict the IRE-induced cell necrosis. Three-dimensionally (3-D) cultured cells in a tissue phantom were experimentally subjected to the electric pulses through a pair of puncture electrodes. The necrotized area was determined as a function of the pulse repetition and compared with the area that was estimated by the numerical analysis.

  9. "Vascular lock" causing splenic perfusion defects during irreversible electroporation of a locally advanced pancreatic tumor.

    Science.gov (United States)

    Ierardi, Anna Maria; Lucchina, Natalie; Duka, Ejona; Bacuzzi, Alessandro; Dionigi, Gianlorenzo; Carrafiello, Gianpaolo

    2014-11-28

    There is little reported experience of irreversible electroporation (IRE) of locally advanced pancreatic tumors (LAP). In literature, few data reported complications. In particular vascular vasoconstriction miming splenic infarcts in humans has never been found. This report describes the onset of asymptomatic multiple little splenic perfusion defects after the treatment of a LAP localized in the boby-tail portion of the pancreas with the application of five percutaneous probes for IRE, in a 79 year-old man. Splenic artery was regularly patent but entirely trapped in the tumor. To the best of our knowledge, until now, no experience concerning percutaneous IRE of pancreatic cancer described that phenomenon. The cause could not be established with certainty and "vascular lock" may be a valid hypothesis. Additional studies are necessary to evaluate its frequency and its exact pathophysiological cause in humans.

  10. Using non-thermal irreversible electroporation to create an in vivo niche for exogenous cell engraftment.

    Science.gov (United States)

    Chang, Tammy T; Zhou, Vivian X; Rubinsky, Boris

    2017-05-01

    The critical shortage of donor organs has spurred investigation of alternative approaches to either generate replacement organs or implant exogenous cells for treatment of end-stage organ failure. Non-thermal irreversible electroporation (NTIRE), which uses brief high electric field pulses to induce irreversible permeabilization of cell membranes, has emerged as a technique for tumor ablation. Here, we introduce a new application for NTIRE that employs in situ cell ablation to create a niche within a solid organ for engraftment of exogenous cells in vivo. We treated the livers of mice with NTIRE and subsequently implanted exogenous congenic hepatocytes within the zone of cell ablation. Donor hepatocytes engrafted and integrated with host liver parenchyma pre-treated with NTIRE. This new approach should have value for studying the effects of a native matrix scaffold on in vivo cell growth and may pioneer a new type of minimally-invasive regenerative surgery.

  11. An Effective Strategy, Applicable to Streptococcus salivarius and Related Bacteria, To Enhance or Confer Electroporation Competence

    Science.gov (United States)

    Buckley, Nicole D.; Vadeboncoeur, Christian; LeBlanc, Donald J.; Lee, Linda N.; Frenette, Michel

    1999-01-01

    Despite the large number of techniques available for transformation of bacteria, certain species and strains are still resistant to introduction of foreign DNA. Some oral streptococci are among the organisms that can be particularly difficult to transform. We performed a series of experiments that involved manipulation of growth and recovery media and cell wall weakening, in which the electroporation conditions, cell concentration, and type and concentration of the transforming plasmid were varied. The variables were optimized such that a previously untransformable Streptococcus salivarius strain, ATCC 25975, could be transformed reproducibly at a level of 105 transformants per μg of DNA. The technique was used to introduce a plasmid into other strains of S. salivarius, including a fresh isolate. Moreover, the same technique was applied successfully to a wide range of oral streptococci and other gram-positive bacteria. PMID:10473378

  12. Irreversible Electroporation of Hepatic and Pancreatic Malignancies: Radiologic-Pathologic Correlation.

    Science.gov (United States)

    Gonzalez-Beicos, Aldo; Venkat, Shree; Songrug, Tanakorn; Poveda, Julio; Garcia-Buitrago, Monica; Poozhikunnath Mohan, Prasoon; Narayanan, Govindarajan

    2015-09-01

    Irreversible electroporation (IRE) is a novel therapy that has shown to be a feasible and promising alternative to conventional ablative techniques when treating tumors near vital structures or blood vessels. The clinical efficacy of IRE has been evaluated using established imaging criteria. This study evaluates the histologic and imaging response of hepatic and pancreatic malignancies that were surgically resected after IRE. In total, 12 lesions ablated with IRE were included, including 3 pancreatic carcinomas, 5 primary tumors of the liver, and 4 metastatic tumors of the liver. The rate of complete response to IRE was 25% based on the histologic evaluation of the resected tumors. Although treatment-related vessel wall changes were noted in several cases in histologic findings, there was no evidence of vascular luminal narrowing or obliteration in any of the specimens. The imaging response to IRE before surgical resection usually resulted in underestimation of disease burden when compared with the histologic response seen on the resected specimens.

  13. Electroporation Transfection as an Effective Tool to Trace Transplanted NSCs in Adult Central Nervous System

    Institute of Scientific and Technical Information of China (English)

    周畅; 温哲钘; 王志萍; 郭行; 史冬梅; 左焕琮; 谢佐平

    2004-01-01

    Neural stem cells, which are clonogenic cells with self-renewal and multilineage differentiation properties, are currently considered as powerful candidates for cell replacement therapy in neurodegenerative disorders such as Parkinson's disease. A key issue is whether stem cells can survive, migrate and differentiate following transplantation into the adult central nervous system. This research shows that enhanced green fluorescent protein (EGFP) plasmid electroporation transfected neural stem cells can functionally differentiate in vitro and that most of the EGFP-positive cells can survive and migrate towards the damaged areas when transplanted into the brain of a Parkinson's disease model rat. The results suggest an effective and maneuverable tracing tool to detect whether transplanted neural stem and progenitor cells function in the adult brain in vivo.

  14. Pretreatment of excess sludge by electroporation; Vorbehandlung von Ueberschussschlamm durch Elektroporation

    Energy Technology Data Exchange (ETDEWEB)

    Kopplow, O.; Barjenbruch, M. [Univ. Rostock, Rostock (Germany); Heinz, V. [TU Berlin, Berlin (Germany)

    2003-07-01

    Disintegration of sewage sludge destroys microorganisms and releases cell constituents, thus making carbon available more quickly for reactions. Electroporation is a high-power pulsed technology which has hardly ever been tested in Germany. The paper presents laboratory results, which are compared with other processes (high-pressure homogeniser, thermal treatment). (orig.) [German] Der anaerobe Stabilisationsprozess haengt unter anderem von der Bioverfuegbarkeit des Kohlenstoffs ab. Durch eine Vorbehandlung (Desintegration) des Schlammes kann infolge der Zerstoerung von Mikroorganismen und der Freisetzung von Zellinhaltsstoffen der Kohlenstoff mikrobiell besser und schneller umgesetzt werden. Die Elektroporation, eine Technologie der Hochleistungspulstechnik, wird als Desintegrationsverfahren in der Literatur genannt, es liegen aber kaum Ausfuehrungen dazu in Deutschland vor. Erste Ergebnisse, die in Laborversuchen mit der Elektroporation von Ueberschussschlamm gewonnen wurden, werden hier vorgestellt und mit anderer Verfahren (Hochdruckhomogenisator, thermische Behandlung) verglichen. (orig.)

  15. Fast gene transfer into the adult zebrafish brain by herpes simplex virus 1 (HSV-1 and electroporation: methods and optogenetic applications

    Directory of Open Access Journals (Sweden)

    Ming eZou

    2014-05-01

    Full Text Available The zebrafish has various advantages as a model organism to analyze the structure and function of neural circuits but efficient viruses or other tools for fast gene transfer are lacking. We show that transgenes can be introduced directly into the adult zebrafish brain by herpes simplex type I viruses (HSV-1 or electroporation. We developed a new procedure to target electroporation to defined brain areas and identified promoters that produced strong long-term expression. The fast workflow of electroporation was exploited to express multiple channelrhodopsin-2 variants and genetically encoded calcium indicators in telencephalic neurons for measurements of neuronal activity and synaptic connectivity. The results demonstrate that HSV-1 and targeted electroporation are efficient tools for gene delivery into the zebrafish brain, similar to adeno-associated viruses and lentiviruses in other species. These methods fill an important gap in the spectrum of molecular tools for zebrafish and are likely to have a wide range of applications.

  16. Profile control studies for JET optimised shear regime

    Energy Technology Data Exchange (ETDEWEB)

    Litaudon, X.; Becoulet, A.; Eriksson, L.G.; Fuchs, V.; Huysmans, G.; How, J.; Moreau, D.; Rochard, F.; Tresset, G.; Zwingmann, W. [Association Euratom-CEA, CEA/Cadarache, Dept. de Recherches sur la Fusion Controlee, DRFC, 13 - Saint-Paul-lez-Durance (France); Bayetti, P.; Joffrin, E.; Maget, P.; Mayorat, M.L.; Mazon, D.; Sarazin, Y. [JET Abingdon, Oxfordshire (United Kingdom); Voitsekhovitch, I. [Universite de Provence, LPIIM, Aix-Marseille 1, 13 (France)

    2000-03-01

    This report summarises the profile control studies, i.e. preparation and analysis of JET Optimised Shear plasmas, carried out during the year 1999 within the framework of the Task-Agreement (RF/CEA/02) between JET and the Association Euratom-CEA/Cadarache. We report on our participation in the preparation of the JET Optimised Shear experiments together with their comprehensive analyses and the modelling. Emphasis is put on the various aspects of pressure profile control (core and edge pressure) together with detailed studies of current profile control by non-inductive means, in the prospects of achieving steady, high performance, Optimised Shear plasmas. (authors)

  17. Application of optimisation techniques in groundwater quantity and quality management

    Indian Academy of Sciences (India)

    Amlan Das; Bithin Datta

    2001-08-01

    This paper presents the state-of-the-art on application of optimisation techniques in groundwater quality and quantity management. In order to solve optimisation-based groundwater management models, researchers have used various mathematical programming techniques such as linear programming (LP), nonlinear programming (NLP), mixed-integer programming (MIP), optimal control theory-based mathematical programming, differential dynamic programming (DDP), stochastic programming (SP), combinatorial optimisation (CO), and multiple objective programming for multipurpose management. Studies reported in the literature on the application of these methods are reviewed in this paper.

  18. Genetic Algorithm Optimisation of a Ship Navigation System

    Directory of Open Access Journals (Sweden)

    E. Alfaro-Cid

    2001-01-01

    Full Text Available The optimisation of the PID controllers' gains for separate propulsion and heading control systems of CyberShip I, a scale model of an oil platform supply ship, using Genetic Algorithms is considered. During the initial design process both PID controllers have been manually tuned to improve their performance. However this tuning approach is a tedious and time consuming process. A solution to this problem is the use of optimisation techniques based on Genetic Algorithms to optimise the controllers' gain values. This investigation has been carried out through computer-generated simulations based on a non-linear hydrodynamic model of CyberShip I.

  19. Towards the creation of decellularized organ constructs using irreversible electroporation and active mechanical perfusion

    Directory of Open Access Journals (Sweden)

    Robertson John

    2010-12-01

    Full Text Available Abstract Background Despite advances in transplant surgery and general medicine, the number of patients awaiting transplant organs continues to grow, while the supply of organs does not. This work outlines a method of organ decellularization using non-thermal irreversible electroporation (N-TIRE which, in combination with reseeding, may help supplement the supply of organs for transplant. Methods In our study, brief but intense electric pulses were applied to porcine livers while under active low temperature cardio-emulation perfusion. Histological analysis and lesion measurements were used to determine the effects of the pulses in decellularizing the livers as a first step towards the development of extracellular scaffolds that may be used with stem cell reseeding. A dynamic conductivity numerical model was developed to simulate the treatment parameters used and determine an irreversible electroporation threshold. Results Ninety-nine individual 1000 V/cm 100-μs square pulses with repetition rates between 0.25 and 4 Hz were found to produce a lesion within 24 hours post-treatment. The livers maintained intact bile ducts and vascular structures while demonstrating hepatocytic cord disruption and cell delamination from cord basal laminae after 24 hours of perfusion. A numerical model found an electric field threshold of 423 V/cm under specific experimental conditions, which may be used in the future to plan treatments for the decellularization of entire organs. Analysis of the pulse repetition rate shows that the largest treated area and the lowest interstitial density score was achieved for a pulse frequency of 1 Hz. After 24 hours of perfusion, a maximum density score reduction of 58.5 percent had been achieved. Conclusions This method is the first effort towards creating decellularized tissue scaffolds that could be used for organ transplantation using N-TIRE. In addition, it provides a versatile platform to study the effects of pulse

  20. Asymmetric Waveforms Decrease Lethal Thresholds in High Frequency Irreversible Electroporation Therapies

    Science.gov (United States)

    Sano, Michael B.; Fan, Richard E.; Xing, Lei

    2017-01-01

    Irreversible electroporation (IRE) is a promising non-thermal treatment for inoperable tumors which uses short (50–100 μs) high voltage monopolar pulses to disrupt the membranes of cells within a well-defined volume. Challenges with IRE include complex treatment planning and the induction of intense muscle contractions. High frequency IRE (H-FIRE) uses bursts of ultrashort (0.25–5 μs) alternating polarity pulses to produce more predictable ablations and alleviate muscle contractions associated with IRE. However, H-FIRE generally ablates smaller volumes of tissue than IRE. This study shows that asymmetric H-FIRE waveforms can be used to create ablation volumes equivalent to standard IRE treatments. Lethal thresholds (LT) of 505 V/cm and 1316 V/cm were found for brain cancer cells when 100 μs IRE and 2 μs symmetric H-FIRE waveforms were used. In contrast, LT as low as 536 V/cm were found for 2 μs asymmetric H-FIRE waveforms. Reversible electroporation thresholds were 54% lower than LTs for symmetric waveforms and 33% lower for asymmetric waveforms indicating that waveform symmetry can be used to tune the relative sizes of reversible and irreversible ablation zones. Numerical simulations predicted that asymmetric H-FIRE waveforms are capable of producing ablation volumes which were 5.8–6.3x larger than symmetric H-FIRE waveforms indicating that in vivo investigation of asymmetric waveforms is warranted.

  1. Achieving magneto-elasto-electroporation and cell transport using core-shell magnetoelectric nanoparticles (Conference Presentation)

    Science.gov (United States)

    Betal, Soutik; Dutta, Moumita; Shrestha, Binita; Saha, Amit; Tang, Liang; Ramasubramanian, Ananad K.; Bhalla, Amar S.; Guo, Ruyan

    2016-09-01

    Magneto-Elasto-Electroporation (MEEP) is a magnetically controlled acoustic-electroporation observed while core-shell Magneto-electric nanoparticles interact with Biological Cells. The surface polarity change of the piezoelectric coating (BaTiO3) due to absorption of pressure created due magneto-striction of core (CoFe2O4) in AC magnetic field results in electric field (Uext) change at the external vicinity of the cell membrane when nanoparticles are nearby. This results in transmembrane Voltage (Um) change which is basically the difference in Cell's internal potential (Uint) and external potential. The nonlinear permeability change of cell membrane due to change in Um opens the nano-pores on the membrane. The magnetic moment of the nanoparticles further helps in penetration of the Magneto-electric nanoparticles inside the cell through these magneto-electrically controlled newly opened nano-pores on cell's membrane. MEEP is analyzed through in-vitro analysis and Mathematical equations are formulated for numerically expressing its fundamental effect. TEM imaging, XRD analysis, Zeta-potentiometer measurement and AFM imaging are confirming the coating of the piezoelectric layer on Magneto-stricitve nanoparticles, Acoustic measurements confirms the photo-acoustic and magneto-acoustic property of CoFe2O4 nanoparticles and Fluorescence microscopy as well as Confocal microscopy are confirming the penetration of particle inside the Human Epithelial cells (HEP2). Further on application of repulsive magnetic field, nanoparticles are observed to transport a group of cells in controlled boundary conditions in microfluidic chamber. Hence these nanoparticles can be used for accurate and efficient drug delivery as well as cell transport applications

  2. Design optimisation of a flywheel hybrid vehicle

    Energy Technology Data Exchange (ETDEWEB)

    Kok, D.B.

    1999-11-04

    This thesis describes the design optimisation of a flywheel hybrid vehicle with respect to fuel consumption and exhaust gas emissions. The driveline of this passenger car uses two power sources: a small spark ignition internal combustion engine with three-way catalyst, and a highspeed flywheel system for kinetic energy storage. A custom-made continuously variable transmission (CVT) with so-called i{sup 2} control transports energy between these power sources and the vehicle wheels. The driveline includes auxiliary systems for hydraulic, vacuum and electric purposes. In this fully mechanical driveline, parasitic energy losses determine the vehicle's fuel saving potential to a large extent. Practicable energy loss models have been derived to quantify friction losses in bearings, gearwheels, the CVT, clutches and dynamic seals. In addition, the aerodynamic drag in the flywheel system and power consumption of auxiliaries are charted. With the energy loss models available, a calculation procedure is introduced to optimise the flywheel as a subsystem in which the rotor geometry, the safety containment, and the vacuum system are designed for minimum energy use within the context of automotive applications. A first prototype of the flywheel system was tested experimentally and subsequently redesigned to improve rotordynamics and safety aspects. Coast-down experiments with the improved version show that the energy losses have been lowered significantly. The use of a kinetic energy storage device enables the uncoupling of vehicle wheel power and engine power. Therefore, the engine can be smaller and it can be chosen to operate in its region of best efficiency in start-stop mode. On a test-rig, the measured engine fuel consumption was reduced with more than 30 percent when the engine is intermittently restarted with the aid of the flywheel system. Although the start-stop mode proves to be advantageous for fuel consumption, exhaust gas emissions increase temporarily

  3. Operational optimisation of water supply networks using a fuzzy ...

    African Journals Online (AJOL)

    Operational optimisation of water supply networks using a fuzzy system. ... This paper presents a fuzzy system to control the pressure in a water distribution network, by using valves and controlling the rotor speed of the ... Article Metrics.

  4. Exploring RSSI Dependency on Height in UHF for throughput optimisation

    CSIR Research Space (South Africa)

    Maliwatu, R

    2016-11-01

    Full Text Available International Conference on Advances in Computing & Communication Engineering (ICACCE), 28-29 November 2016, Durban, South Africa Exploring RSSI Dependency on Height in UHF for throughput optimisation Richard Maliwatu Albert Lysko David Johnson...

  5. Efficient topology optimisation of multiscale and multiphysics problems

    DEFF Research Database (Denmark)

    Alexandersen, Joe

    The aim of this Thesis is to present efficient methods for optimising high-resolution problems of a multiscale and multiphysics nature. The Thesis consists of two parts: one treating topology optimisation of microstructural details and the other treating topology optimisation of conjugate heat...... transfer problems. Part I begins with an introduction to the concept of microstructural details in the context of topology optimisation. Relevant literature is briefly reviewed and problems with existing methodologies are identified. The proposed methodology and its strengths are summarised. Details...... the computational cost of treating structures with fully-resolved microstructural details. The methodology is further applied to examples, where it is shown that it ensures connectivity of the microstructural details and that forced periodicity of the microstructural details can yield an implicit robustness to load...

  6. Share-of-Surplus Product Line Optimisation with Price Levels

    Directory of Open Access Journals (Sweden)

    X. G. Luo

    2014-01-01

    Full Text Available Kraus and Yano (2003 established the share-of-surplus product line optimisation model and developed a heuristic procedure for this nonlinear mixed-integer optimisation model. In their model, price of a product is defined as a continuous decision variable. However, because product line optimisation is a planning process in the early stage of product development, pricing decisions usually are not very precise. In this research, a nonlinear integer programming share-of-surplus product line optimization model that allows the selection of candidate price levels for products is established. The model is further transformed into an equivalent linear mixed-integer optimisation model by applying linearisation techniques. Experimental results in different market scenarios show that the computation time of the transformed model is much less than that of the original model.

  7. Open Pit Optimisation and Design: A Stepwise Approach*

    African Journals Online (AJOL)

    Michael

    2015-12-02

    Dec 2, 2015 ... holes were used for the analysis. ... retrieval and analysis, using Surpac software. .... economic and technical parameters were used to produce a set of nested pits. Fig. 4 depicts a summarised flow chart for the pit optimisation.

  8. Optimisation of patient protection and image quality in diagnostic ...

    African Journals Online (AJOL)

    Optimisation of patient protection and image quality in diagnostic radiology. ... The study leads to the introduction of the concept of plan- do-check-act on QC results ... (QA) programme and continues to collect data for establishment of DRL's.

  9. A Comparison of Existing Optimisation Techniques with the ...

    African Journals Online (AJOL)

    ... Existing Optimisation Techniques with the Univariate Marginal Distribution Algorithm ... graph colouring, neural networks, genetic algorithms and tabu search. ... optimization techniques and we show how the proposed algorithm performs in ...

  10. Elliptical Antenna Array Synthesis Using Backtracking Search Optimisation Algorithm

    Directory of Open Access Journals (Sweden)

    Kerim Guney

    2016-04-01

    Full Text Available The design of the elliptical antenna arrays is relatively new research area in the antenna array community. Backtracking search optimisation algorithm (BSA is employed for the synthesis of elliptical antenna arrays having different number of array elements. For this aim, BSA is used to calculate the optimum angular position and amplitude values of the array elements. BSA is a population-based iterative evolutionary algorithm. The remarkable properties of BSA are that it has a good optimisation performance, simple implementation structure, and few control parameters. The results of BSA are compared with those of self-adaptive differential evolution algorithm, firefly algorithm, biogeography based optimisation algorithm, and genetic algorithm. The results show that BSA can reach better solutions than the compared optimisation algorithms. Iterative performances of BSA are also compared with those of bacterial foraging algorithm and differential search algorithm.

  11. Construction and optimisation of a cartridge filter for removing ...

    African Journals Online (AJOL)

    Construction and optimisation of a cartridge filter for removing fluoride in drinking water. ... It was found that the optimal conditions for the F- filter that gave the best results in removing of F- from water with minimum ... Article Metrics.

  12. Weight Optimisation of Steel Monopile Foundations for Offshore Windfarms

    DEFF Research Database (Denmark)

    Fog Gjersøe, Nils; Bouvin Pedersen, Erik; Kristensen, Brian;

    2015-01-01

    The potential for mass reduction of monopiles in offshore windfarms using current design practice is investigated. Optimisation by sensitivity analysis is carried out for the following important parameters: wall thickness distribution between tower and monopile, soil stiffness, damping ratio and ...

  13. optPBN: An Optimisation Toolbox for Probabilistic Boolean Networks

    Science.gov (United States)

    Trairatphisan, Panuwat; Mizera, Andrzej; Pang, Jun; Tantar, Alexandru Adrian; Sauter, Thomas

    2014-01-01

    Background There exist several computational tools which allow for the optimisation and inference of biological networks using a Boolean formalism. Nevertheless, the results from such tools yield only limited quantitative insights into the complexity of biological systems because of the inherited qualitative nature of Boolean networks. Results We introduce optPBN, a Matlab-based toolbox for the optimisation of probabilistic Boolean networks (PBN) which operates under the framework of the BN/PBN toolbox. optPBN offers an easy generation of probabilistic Boolean networks from rule-based Boolean model specification and it allows for flexible measurement data integration from multiple experiments. Subsequently, optPBN generates integrated optimisation problems which can be solved by various optimisers. In term of functionalities, optPBN allows for the construction of a probabilistic Boolean network from a given set of potential constitutive Boolean networks by optimising the selection probabilities for these networks so that the resulting PBN fits experimental data. Furthermore, the optPBN pipeline can also be operated on large-scale computational platforms to solve complex optimisation problems. Apart from exemplary case studies which we correctly inferred the original network, we also successfully applied optPBN to study a large-scale Boolean model of apoptosis where it allows identifying the inverse correlation between UVB irradiation, NFκB and Caspase 3 activations, and apoptosis in primary hepatocytes quantitatively. Also, the results from optPBN help elucidating the relevancy of crosstalk interactions in the apoptotic network. Summary The optPBN toolbox provides a simple yet comprehensive pipeline for integrated optimisation problem generation in the PBN formalism that can readily be solved by various optimisers on local or grid-based computational platforms. optPBN can be further applied to various biological studies such as the inference of gene regulatory

  14. A supportive architecture for CFD-based design optimisation

    Science.gov (United States)

    Li, Ni; Su, Zeya; Bi, Zhuming; Tian, Chao; Ren, Zhiming; Gong, Guanghong

    2014-03-01

    Multi-disciplinary design optimisation (MDO) is one of critical methodologies to the implementation of enterprise systems (ES). MDO requiring the analysis of fluid dynamics raises a special challenge due to its extremely intensive computation. The rapid development of computational fluid dynamic (CFD) technique has caused a rise of its applications in various fields. Especially for the exterior designs of vehicles, CFD has become one of the three main design tools comparable to analytical approaches and wind tunnel experiments. CFD-based design optimisation is an effective way to achieve the desired performance under the given constraints. However, due to the complexity of CFD, integrating with CFD analysis in an intelligent optimisation algorithm is not straightforward. It is a challenge to solve a CFD-based design problem, which is usually with high dimensions, and multiple objectives and constraints. It is desirable to have an integrated architecture for CFD-based design optimisation. However, our review on existing works has found that very few researchers have studied on the assistive tools to facilitate CFD-based design optimisation. In the paper, a multi-layer architecture and a general procedure are proposed to integrate different CFD toolsets with intelligent optimisation algorithms, parallel computing technique and other techniques for efficient computation. In the proposed architecture, the integration is performed either at the code level or data level to fully utilise the capabilities of different assistive tools. Two intelligent algorithms are developed and embedded with parallel computing. These algorithms, together with the supportive architecture, lay a solid foundation for various applications of CFD-based design optimisation. To illustrate the effectiveness of the proposed architecture and algorithms, the case studies on aerodynamic shape design of a hypersonic cruising vehicle are provided, and the result has shown that the proposed architecture

  15. Multiobjective optimisation of bogie suspension to boost speed on curves

    Science.gov (United States)

    Milad Mousavi-Bideleh, Seyed; Berbyuk, Viktor

    2016-01-01

    To improve safety and maximum admissible speed on different operational scenarios, multiobjective optimisation of bogie suspension components of a one-car railway vehicle model is considered. The vehicle model has 50 degrees of freedom and is developed in multibody dynamics software SIMPACK. Track shift force, running stability, and risk of derailment are selected as safety objective functions. The improved maximum admissible speeds of the vehicle on curves are determined based on the track plane accelerations up to 1.5 m/s2. To attenuate the number of design parameters for optimisation and improve the computational efficiency, a global sensitivity analysis is accomplished using the multiplicative dimensional reduction method (M-DRM). A multistep optimisation routine based on genetic algorithm (GA) and MATLAB/SIMPACK co-simulation is executed at three levels. The bogie conventional secondary and primary suspension components are chosen as the design parameters in the first two steps, respectively. In the last step semi-active suspension is in focus. The input electrical current to magnetorheological yaw dampers is optimised to guarantee an appropriate safety level. Semi-active controllers are also applied and the respective effects on bogie dynamics are explored. The safety Pareto optimised results are compared with those associated with in-service values. The global sensitivity analysis and multistep approach significantly reduced the number of design parameters and improved the computational efficiency of the optimisation. Furthermore, using the optimised values of design parameters give the possibility to run the vehicle up to 13% faster on curves while a satisfactory safety level is guaranteed. The results obtained can be used in Pareto optimisation and active bogie suspension design problems.

  16. Optimisation as a process for understanding and managing river ecosystems

    OpenAIRE

    Barbour, EJ; Holz, L; G. Kuczera; Pollino, CA; Jakeman, AJ; Loucks, DP

    2016-01-01

    Optimisation can assist in the management of riverine ecosystems through the exploration of multiple alternative management strategies, and the evaluation of trade-offs between conflicting objectives. In addition, it can facilitate communication and learning about the system. However, the effectiveness of optimisation in aiding decision making for ecological management is currently limited by four major challenges: identification and quantification of ecosystem objectives; representation of e...

  17. Optimisation of the Sekwa blended-wing-Body research UAV

    CSIR Research Space (South Africa)

    Broughton, BA

    2008-10-01

    Full Text Available candidate design to bring the total mass up to the target total mass of 3.2 kg. The location of the ballast mass could be adjusted by the design code, which allowed the static margin to be used as a design variable. Finally, a series of checks were.... Overview of optimisation process OPTIMISER Genetic Algorithms + Gradient Based Methods Natural FQ constraints Geometric constraints Control system constraints Stall behaviour Design with best cruise performance Design parameters Generate...

  18. Aerodynamic optimisation of an industrial axial fan blade

    OpenAIRE

    2006-01-01

    Numerical optimisation methods have successfully been used for a variety of aerodynamic design problems over quite a few years. However the application of these methods to the aerodynamic blade shape optimisation of industrial axial fans has received much less attention in the literature probably given the fact that the majority of resources available to develop these automated design approaches is to be found in the aerospace field. This work presents the develo...

  19. Spreadsheets In Function Of Optimisation Of Logistics Network

    OpenAIRE

    Drago Pupavac; Mimo Draskovic

    2007-01-01

    This scientific paper discusses how estimated spreadsheets functions in logistics networks optimisation. The suggested working hypothesis for efficacy of estimated spreadsheets in designing logistics networks is proved and a practical example. In this way the given model can be applied to all logistics networks of similar problem capacity. Logistics network model confronting estimated spreadsheets present a real world at a level needed for understanding the problem of optimisation of logistic...

  20. Optimisation study of a vehicle bumper subsystem with fuzzy parameters

    Science.gov (United States)

    Farkas, L.; Moens, D.; Donders, S.; Vandepitte, D.

    2012-10-01

    This paper deals with the design and optimisation for crashworthiness of a vehicle bumper subsystem, which is a key scenario for vehicle component design. The automotive manufacturers and suppliers have to find optimal design solutions for such subsystems that comply with the conflicting requirements of the regulatory bodies regarding functional performance (safety and repairability) and regarding the environmental impact (mass). For the bumper design challenge, an integrated methodology for multi-attribute design engineering of mechanical structures is set up. The integrated process captures the various tasks that are usually performed manually, this way facilitating the automated design iterations for optimisation. Subsequently, an optimisation process is applied that takes the effect of parametric uncertainties into account, such that the system level of failure possibility is acceptable. This optimisation process is referred to as possibility-based design optimisation and integrates the fuzzy FE analysis applied for the uncertainty treatment in crash simulations. This process is the counterpart of the reliability-based design optimisation used in a probabilistic context with statistically defined parameters (variabilities).

  1. The Electrostatic Component of the Disjoining Pressure and the Pore Creation Rate in Electroporation Models and Theory

    CERN Document Server

    Vasilkoski, Zlatko

    2008-01-01

    Under externally applied electric fields, lipid membranes tend to permeate and change their electrical resistance by the combined processes of pore creation and pore evolution (expansion or contraction). This study is focused on the pore creation process, represented by an empirical expression currently used in the electroporation (EP) models, for which an alternative theoretically based expression was provided. The choice of this expression was motivated by the role the DLVO's (disjoining) pressures may play in the process of EP. The electrostatic energy effects on each sides of a lipid membrane were evaluated in terms of the electrostatic component of the disjoining pressure. Thus the pore creation energy considerations in the current EP models, associated with the necessity of an idealized non conducting circular pre-pore were avoided. As a result, a new expression for the onset of the electroporation was proposed. It was found that this new theoretically determined expression is in good agreement with the...

  2. Optimised control of coal-fired boilers

    Energy Technology Data Exchange (ETDEWEB)

    Owens, D.H.; MacConnell, P.F.A.; Neuffer, D.; Dando, R. [University of Exeter, Exeter (United Kingdom). Centre for System and Control Engineering

    1997-07-01

    The objective of the project is to develop and specify a control methodology that will enable existing coal combustion plant to take maximum advantage of modern control techniques. The research is specifically aimed at chain-grate stoker plant (such as the test facility at the Coal Research Establishment, Cheltenham) on which little work has been done for thirty years yet which still represents a large proportion of industrial coal-fired plant in operation worldwide. In detail, the project: reviewed existing control strategies for moving grate stokers, highlighting their limitations and areas for improvements; carried out plant trials to identify the system characteristics such as response time and input/output behaviour; developed a theoretical process based on physical and chemical laws and backed up by trial data; specified control strategies for a single boiler; simulated and evaluated the control strategies using model simulations; developed of an optimised. Control strategy for a single boiler; and assessed the applicability and effects of this control strategy on multiple boiler installations. 67 refs., 34 figs.

  3. Semantic Query Optimisation with Ontology Simulation

    CERN Document Server

    Gupta, Siddharth

    2010-01-01

    Semantic Web is, without a doubt, gaining momentum in both industry and academia. The word "Semantic" refers to "meaning" - a semantic web is a web of meaning. In this fast changing and result oriented practical world, gone are the days where an individual had to struggle for finding information on the Internet where knowledge management was the major issue. The semantic web has a vision of linking, integrating and analysing data from various data sources and forming a new information stream, hence a web of databases connected with each other and machines interacting with other machines to yield results which are user oriented and accurate. With the emergence of Semantic Web framework the na\\"ive approach of searching information on the syntactic web is clich\\'e. This paper proposes an optimised semantic searching of keywords exemplified by simulation an ontology of Indian universities with a proposed algorithm which ramifies the effective semantic retrieval of information which is easy to access and time sav...

  4. ENERGY OPTIMISATION SCHEMES FOR WIRELESS SENSOR NETWORK

    Directory of Open Access Journals (Sweden)

    Vivekanand Jha

    2012-05-01

    Full Text Available A sensor network is composed of a large number of sensor nodes, which are densely deployed either inside the phenomenon or very close to it. Sensor nodes have sensing, processing and transmitting capability . They however have limited energy and measures need to be taken to make op- timum usage of their energy and save them from task of only receiving and transmitting data without processing. Various techniques for energy utilization optimisation have been proposed Ma jor players are however clustering and relay node placement. In the research related to relay node placement, it has been proposed to deploy some relay nodes such that the sensors can transmit the sensed data to a nearby relay node, which in turn delivers the data to the base stations. In general, the relay node placement problems aim to meet certain connectivity and/or survivabil- ity requirements of the network by deploying a minimum number of relay nodes. The other approach is grouping sensor nodes into clusters with each cluster having a cluster head (CH. The CH nodes aggregate the data and transmit them to the base station (BS. These two approaches has been widely adopted by the research community to satisfy the scala- bility objective and generally achieve high energy efficiency and prolong network lifetime in large-scale WSN environments and hence are discussed here along with single hop and multi hop characteristic of sensor node.

  5. Topological optimisation of rod-stirring devices

    CERN Document Server

    Finn, Matthew D

    2011-01-01

    There are many industrial situations where rods are used to stir a fluid, or where rods repeatedly stretch a material such as bread dough or taffy. The goal in these applications is to stretch either material lines (in a fluid) or the material itself (for dough or taffy) as rapidly as possible. The growth rate of material lines is conveniently given by the topological entropy of the rod motion. We discuss the problem of optimising such rod devices from a topological viewpoint. We express rod motions in terms of generators of the braid group, and assign a cost based on the minimum number of generators needed to write the braid. We show that for one cost function -- the topological entropy per generator -- the optimal growth rate is the logarithm of the golden ratio. For a more realistic cost function,involving the topological entropy per operation where rods are allowed to move together, the optimal growth rate is the logarithm of the silver ratio, $1+\\sqrt{2}$. We show how to construct devices that realise th...

  6. Optimising preterm nutrition: present and future

    LENUS (Irish Health Repository)

    Brennan, Ann-Marie

    2016-04-01

    The goal of preterm nutrition in achieving growth and body composition approximating that of the fetus of the same postmenstrual age is difficult to achieve. Current nutrition recommendations depend largely on expert opinion, due to lack of evidence, and are primarily birth weight based, with no consideration given to gestational age and\\/or need for catch-up growth. Assessment of growth is based predominately on anthropometry, which gives insufficient attention to the quality of growth. The present paper provides a review of the current literature on the nutritional management and assessment of growth in preterm infants. It explores several approaches that may be required to optimise nutrient intakes in preterm infants, such as personalising nutritional support, collection of nutrient intake data in real-time, and measurement of body composition. In clinical practice, the response to inappropriate nutrient intakes is delayed as the effects of under- or overnutrition are not immediate, and there is limited nutritional feedback at the cot-side. The accurate and non-invasive measurement of infant body composition, assessed by means of air displacement plethysmography, has been shown to be useful in assessing quality of growth. The development and implementation of personalised, responsive nutritional management of preterm infants, utilising real-time nutrient intake data collection, with ongoing nutritional assessments that include measurement of body composition is required to help meet the individual needs of preterm infants.

  7. Electroporation and lipid nanoparticles with cyanine IR-780 and flavonoids as efficient vectors to enhanced drug delivery in colon cancer.

    Science.gov (United States)

    Kulbacka, Julita; Pucek, Agata; Kotulska, Małgorzata; Dubińska-Magiera, Magda; Rossowska, Joanna; Rols, Marie-Pierre; Wilk, Kazimiera Anna

    2016-08-01

    Nanocarriers and electroporation (also named electropermeabilization) are convenient methods to increase drug transport. In the current study, we present an effective support of drug delivery into cancer cells, utilizing these methods. We compare the efficiency of each of them and their combination. Multifunctional solid lipid nanoparticles (SLNs) loaded with a cyanine-type IR-780 - acting as a diagnostic agent and a photosensitizer, and a flavonoid derivative - baicalein (BAI) or fisetin (FIS) as a therapeutic cargo - were fabricated via solvent-diffusion method. A therapy supplemented with flavonoids may provide a more precise method to apply desirable lower drug doses and is more likely to result in lower toxicity and a decrease in tumor growth. The SLNs were stabilized with Phospholipon 90G at various concentrations; cetyl palmitate (CP) was applied as a solid matrix. The obtained nanosystems were characterized by dynamic light scattering (size along with size distribution), UV-vis (cargos encapsulation efficiency) and atomic force microscopy (morphology and shape). The obtained SLNs were used as drug carriers alone and in combination with electropermeabilization induced by millisecond pulsed electric fields of high intensity. Two cell lines were selected for the study: LoVo and CHO-K1. The viability was assessed after electroporation alone, the use of electroporation and nanoparticles, and nanoparticles or drugs alone. The intracellular accumulation of cyanine IR-780 and the impact on intracellular structure organization of cytoskeleton was visualized with confocal microscopy method with alpha-actin and beta-tubulin. In this study, the efficacy of nanoparticles with mixed cargo, additionally enhanced by electroporation, is demonstrated to act as an anticancer modality to eliminate cancer cells.

  8. Targeted Collection of Plasmid DNA in Large and Growing Animal Muscles 6 Weeks after DNA Vaccination with and without Electroporation

    Directory of Open Access Journals (Sweden)

    Daniel Dory

    2015-01-01

    Full Text Available DNA vaccination has been developed in the last two decades in human and animal species as a promising alternative to conventional vaccination. It consists in the injection, in the muscle, for example, of plasmid DNA encoding the vaccinating polypeptide. Electroporation which forces the entrance of the plasmid DNA in cells at the injection point has been described as a powerful and promising strategy to enhance DNA vaccine efficacy. Due to the fact that the vaccine is composed of DNA, close attention on the fate of the plasmid DNA upon vaccination has to be taken into account, especially at the injection point. To perform such studies, the muscle injection point has to be precisely recovered and collected several weeks after injection. This is even more difficult for large and growing animals. A technique has been developed to localize precisely and collect efficiently the muscle injection points in growing piglets 6 weeks after DNA vaccination accompanied or not by electroporation. Electroporation did not significantly increase the level of remaining plasmids compared to nonelectroporated piglets, and, in all the cases, the levels were below the limit recommended by the FDA to research integration events of plasmid DNA into the host DNA.

  9. BTX AgilePulse(TM) system is an effective electroporation device for intramuscular and intradermal delivery of DNA vaccine.

    Science.gov (United States)

    Davtyan, Hayk; Hovakimyan, Armine; Zagorski, Karen; Davtyan, Arpine; Petrushina, Irina; Agdashian, David; Murthy, Vidya; Cribbs, David H; Agadjanyan, Michael G; Ghochikyan, Anahit

    2014-01-01

    DNA vaccines promote immune system activation in small animals and exhibit certain advantages when compared to conventional recombinant protein vaccines. However in clinical trials DNA vaccines are less effective in inducing potent immune responses due to the low delivery efficiency and expression of antigens. Currently, various delivery devices such as gene-guns, bioinjectors and electroporation systems are being used in order to increase the potency of DNA vaccines. However, the optimal delivery parameters are required and must be carefully set to obtain the highest levels of gene expression and strong immune responses in humans. The focus of this study was to optimize electroporation settings (voltage, pulse length, pulse intervals, and number of pulses), as well as the route of administration (intradermal vs. intramuscular) and dosage of the DNA epitope vaccine, AV-1959D, delivered by the BTX AgilePulse(TM) system. As a result, we have chosen the optimal settings for electroporation delivery using different routes of immunization with this vaccine, generating (i) robust antibody production to the B cell epitope (a small peptide, derived from β-amyloid), and (ii) strong cellular immune responses to Th epitopes (a small synthetic peptide and eleven peptides from various pathogens) incorporated into DNA vaccine platform.

  10. Tolerability of intramuscular and intradermal delivery by CELLECTRA(®) adaptive constant current electroporation device in healthy volunteers.

    Science.gov (United States)

    Diehl, Malissa C; Lee, Jessica C; Daniels, Stephen E; Tebas, Pablo; Khan, Amir S; Giffear, Mary; Sardesai, Niranjan Y; Bagarazzi, Mark L

    2013-10-01

    DNA vaccines are being developed as a potentially safe and effective immunization platform. However, translation of DNA vaccines into a clinical setting has produced results that have fallen short of those generated in a preclinical setting. Various strategies are being developed to address this lack of potency, including improvements in delivery methods. Electroporation (EP) creates transient increases in cell membrane permeability, thus enhancing DNA uptake and leading to a more robust immune response. Here, we report on the safety and tolerability of delivering sterile saline via intramuscular (IM) or intradermal (ID) injection followed by in vivo electroporation using the CELLECTRA(®) adaptive constant current device in healthy adults from two open-label studies. Pain, as assessed by VAS, was highest immediately after EP but diminishes by about 50% within 5 min. Mean VAS scores appear to correlate with the amount of energy delivered and depth of needle insertion, especially for intramuscular EP. Mean scores did not exceed 7 out of 10 or 3 out of 10 for IM and ID EP, respectively. The majority of adverse events included mild to moderate injection site reactions that resolved within one day. No deaths or serious adverse events were reported during the course of either study. Overall, injection followed by EP with the CELLECTRA(®) device was well-tolerated and no significant safety concerns were identified. These studies support the further development of electroporation as a vaccine delivery method to enhance immunogenicity, particularly for diseases in which traditional vaccination approaches are ineffective.

  11. Single cell electroporation for longitudinal imaging of synaptic structure and function in the adult mouse neocortex in vivo

    Directory of Open Access Journals (Sweden)

    Stephane ePages

    2015-04-01

    Full Text Available Longitudinal imaging studies of neuronal structures in vivo have revealed rich dynamics in dendritic spines and axonal boutons. Spines and boutons are considered to be proxies for synapses. This implies that synapses display similar dynamics. However, spines and boutons do not always bear synapses, some may contain more than one, and dendritic shaft synapses have no clear structural proxies. In addition, synaptic strength is not always accurately revealed by just the size of these structures. Structural and functional dynamics of synapses could be studied more reliably using fluorescent synaptic proteins as markers for size and function. These proteins are often large and possibly interfere with circuit development, which renders them less suitable for conventional transfection or transgenesis methods such as viral vectors, in utero electroporation and germline transgenesis. Single cell electroporation has been shown to be a potential alternative for transfection of recombinant fluorescent proteins in adult cortical neurons. Here we provide proof of principle for the use of single cell electroporation to express and subsequently image fluorescently tagged synaptic proteins over days to weeks in vivo.

  12. Cationic solid-lipid nanoparticles are as efficient as electroporation in DNA vaccination against visceral leishmaniasis in mice.

    Science.gov (United States)

    Saljoughian, N; Zahedifard, F; Doroud, D; Doustdari, F; Vasei, M; Papadopoulou, B; Rafati, S

    2013-12-01

    The use of an appropriate delivery system has recently emerged as a promising approach for the development of effective vaccination against visceral leishmaniasis (VL). Here, we compare two vaccine delivery systems, namely electroporation and cationic solid-lipid nanoparticle (cSLN) formulation, to administer a DNA vaccine harbouring the L. donovani A2 antigen along with L. infantum cysteine proteinases [CPA and CPB without its unusual C-terminal extension (CPB(-CTE) )] and evaluate their potential against L. infantum challenge. Prime-boost administration of the pcDNA-A2-CPA-CPB(-CTE) delivered by either electroporation or cSLN formulation protects BALB/c mice against L. infantum challenge and that protective immunity is associated with high levels of IFN-γ and lower levels of IL-10 production, leading to a strong Th1 immune response. At all time points, the ratio of IFN-γ: IL-10 induced upon restimulation with rA2-rCPA-rCPB and F/T antigens was significantly higher in vaccinated animals. Moreover, Th2-efficient protection was elicited through a high humoral immune response. Nitric oxide production, parasite burden and histopathological analysis were also in concordance with other findings. Overall, these data indicate that similar to the electroporation delivery system, cSLNs as a nanoscale vehicle of Leishmania antigens could improve immune response, hence indicating the promise of these strategies against visceral leishmaniasis.

  13. Electroporation Knows No Boundaries: The Use of Electrostimulation for siRNA Delivery in Cells and Tissues.

    Science.gov (United States)

    Luft, Christin; Ketteler, Robin

    2015-09-01

    The discovery of RNA interference (RNAi) has enabled several breakthrough discoveries in the area of functional genomics. The RNAi technology has emerged as one of the major tools for drug target identification and has been steadily improved to allow gene manipulation in cell lines, tissues, and whole organisms. One of the major hurdles for the use of RNAi in high-throughput screening has been delivery to cells and tissues. Some cell types are refractory to high-efficiency transfection with standard methods such as lipofection or calcium phosphate precipitation and require different means. Electroporation is a powerful and versatile method for delivery of RNA, DNA, peptides, and small molecules into cell lines and primary cells, as well as whole tissues and organisms. Of particular interest is the use of electroporation for delivery of small interfering RNA oligonucleotides and clustered regularly interspaced short palindromic repeats/Cas9 plasmid vectors in high-throughput screening and for therapeutic applications. Here, we will review the use of electroporation in high-throughput screening in cell lines and tissues.

  14. Transfection of isolated rainbow trout, Oncorhynchus mykiss, granulosa cells through chemical transfection and electroporation at 12°C.

    Science.gov (United States)

    Marivin, E; Mourot, B; Loyer, P; Rime, H; Bobe, J; Fostier, A

    2015-09-15

    Over-expression or inhibition of gene expression can be efficiently used to analyse the functions and/or regulation of target genes. Modulation of gene expression can be achieved through transfection of exogenous nucleic acids into target cells. Such techniques require the development of specific protocols to transfect cell cultures with nucleic acids. The aim of this study was to develop a method of transfection suitable for rainbow trout granulosa cells in primary culture. After the isolation of rainbow trout granulosa cells, chemical transfection of cells with a fluorescent morpholino oligonucleotide (MO) was tested using FuGENE HD at 12 °C. Electroporation was also employed to transfect these cells with either a plasmid or MO. Transfection was more efficient using electroporation (with the following settings: 1200 V/40 ms/1p) than chemical transfection, but electroporation by itself was deleterious, resulting in a decrease of the steroidogenic capacity of the cells, measured via estradiol production from its androgenic substrate. The disturbance of cell biology induced by the transfection method per se should be taken into account in data interpretation when investigating the effects of under- or over-expression of candidate genes.

  15. Optimisation of the formulation of a bubble bath by a chemometric approach market segmentation and optimisation.

    Science.gov (United States)

    Marengo, Emilio; Robotti, Elisa; Gennaro, Maria Carla; Bertetto, Mariella

    2003-03-01

    The optimisation of the formulation of a commercial bubble bath was performed by chemometric analysis of Panel Tests results. A first Panel Test was performed to choose the best essence, among four proposed to the consumers; the best essence chosen was used in the revised commercial bubble bath. Afterwards, the effect of changing the amount of four components (the amount of primary surfactant, the essence, the hydratant and the colouring agent) of the bubble bath was studied by a fractional factorial design. The segmentation of the bubble bath market was performed by a second Panel Test, in which the consumers were requested to evaluate the samples coming from the experimental design. The results were then treated by Principal Component Analysis. The market had two segments: people preferring a product with a rich formulation and people preferring a poor product. The final target, i.e. the optimisation of the formulation for each segment, was obtained by the calculation of regression models relating the subjective evaluations given by the Panel and the compositions of the samples. The regression models allowed to identify the best formulations for the two segments ofthe market.

  16. High-frequency irreversible electroporation (H-FIRE for non-thermal ablation without muscle contraction

    Directory of Open Access Journals (Sweden)

    Arena Christopher B

    2011-11-01

    Full Text Available Abstract Background Therapeutic irreversible electroporation (IRE is an emerging technology for the non-thermal ablation of tumors. The technique involves delivering a series of unipolar electric pulses to permanently destabilize the plasma membrane of cancer cells through an increase in transmembrane potential, which leads to the development of a tissue lesion. Clinically, IRE requires the administration of paralytic agents to prevent muscle contractions during treatment that are associated with the delivery of electric pulses. This study shows that by applying high-frequency, bipolar bursts, muscle contractions can be eliminated during IRE without compromising the non-thermal mechanism of cell death. Methods A combination of analytical, numerical, and experimental techniques were performed to investigate high-frequency irreversible electroporation (H-FIRE. A theoretical model for determining transmembrane potential in response to arbitrary electric fields was used to identify optimal burst frequencies and amplitudes for in vivo treatments. A finite element model for predicting thermal damage based on the electric field distribution was used to design non-thermal protocols for in vivo experiments. H-FIRE was applied to the brain of rats, and muscle contractions were quantified via accelerometers placed at the cervicothoracic junction. MRI and histological evaluation was performed post-operatively to assess ablation. Results No visual or tactile evidence of muscle contraction was seen during H-FIRE at 250 kHz or 500 kHz, while all IRE protocols resulted in detectable muscle contractions at the cervicothoracic junction. H-FIRE produced ablative lesions in brain tissue that were characteristic in cellular morphology of non-thermal IRE treatments. Specifically, there was complete uniformity of tissue death within targeted areas, and a sharp transition zone was present between lesioned and normal brain. Conclusions H-FIRE is a feasible technique for

  17. In vivo electroporation of morpholinos into the regenerating adult zebrafish tail fin.

    Science.gov (United States)

    Hyde, David R; Godwin, Alan R; Thummel, Ryan

    2012-03-29

    RNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue. This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

  18. A conceptual optimisation strategy for radiography in a digital environment.

    Science.gov (United States)

    Båth, Magnus; Håkansson, Markus; Hansson, Jonny; Månsson, Lars Gunnar

    2005-01-01

    Using a completely digital environment for the entire imaging process leads to new possibilities for optimisation of radiography since many restrictions of screen/film systems, such as the small dynamic range and the lack of possibilities for image processing, do not apply any longer. However, at the same time these new possibilities lead to a more complicated optimisation process, since more freedom is given to alter parameters. This paper focuses on describing an optimisation strategy that concentrates on taking advantage of the conceptual differences between digital systems and screen/film systems. The strategy can be summarised as: (a) always include the anatomical background during the optimisation, (b) perform all comparisons at a constant effective dose and (c) separate the image display stage from the image collection stage. A three-step process is proposed where the optimal setting of the technique parameters is determined at first, followed by an optimisation of the image processing. In the final step the optimal dose level-given the optimal settings of the image collection and image display stages-is determined.

  19. CT dose optimisation and reduction in osteoarticular disease.

    Science.gov (United States)

    Gervaise, A; Teixeira, P; Villani, N; Lecocq, S; Louis, M; Blum, A

    2013-04-01

    With an improvement in the temporal and spatial resolution, computed tomography (CT) is indicated in the evaluation of a great many osteoarticular diseases. New exploration techniques such as the dynamic CT and CT bone perfusion also provide new indications. However, CT is still an irradiating imaging technique and dose optimisation and reduction remains primordial. In this paper, the authors first present the typical doses delivered during CT in osteoarticular disease. They then discuss the different ways to optimise and reduce these doses by distinguishing the behavioural factors from the technical factors. Among the latter, the optimisation of the milliamps and kilovoltage is indispensable and should be adapted to the type of exploration and the morphotype of each individual. These technical factors also benefit from recent technological evolutions with the distribution of iterative reconstructions. In this way, the dose may be divided by two and provide an image of equal quality. With these dose optimisation and reduction techniques, it is now possible, while maintaining an excellent quality of the image, to obtain low-dose or even very low-dose acquisitions with a dose sometimes similar that of a standard X-ray assessment. Nevertheless, although these technical factors provide a major reduction in the dose delivered, behavioural factors, such as compliance with the indications, remain fundamental. Finally, the authors describe how to optimise and reduce the dose with specific applications in musculoskeletal imaging such as the dynamic CT, CT bone perfusion and dual energy CT.

  20. Hybrid Genetic Algorithm with PSO Effect for Combinatorial Optimisation Problems

    Directory of Open Access Journals (Sweden)

    M. H. Mehta

    2012-12-01

    Full Text Available In engineering field, many problems are hard to solve in some definite interval of time. These problems known as “combinatorial optimisation problems” are of the category NP. These problems are easy to solve in some polynomial time when input size is small but as input size grows problems become toughest to solve in some definite interval of time. Long known conventional methods are not able to solve the problems and thus proper heuristics is necessary. Evolutionary algorithms based on behaviours of different animals and species have been invented and studied for this purpose. Genetic Algorithm is considered a powerful algorithm for solving combinatorial optimisation problems. Genetic algorithms work on these problems mimicking the human genetics. It follows principle of “survival of the fittest” kind of strategy. Particle swarm optimisation is a new evolutionary approach that copies behaviour of swarm in nature. However, neither traditional genetic algorithms nor particle swarm optimisation alone has been completely successful for solving combinatorial optimisation problems. Here a hybrid algorithm is proposed in which strengths of both algorithms are merged and performance of proposed algorithm is compared with simple genetic algorithm. Results show that proposed algorithm works definitely better than the simple genetic algorithm.