Electroporation-delivered transdermal neostigmine in rats: equivalent action to intravenous administration

PubMed Central

Berkó, Szilvia; Szűcs, Kálmán F; Balázs, Boglárka; Csányi, Erzsébet; Varju, Gábor; Sztojkov-Ivanov, Anita; Budai-Szűcs, Mária; Bóta, Judit; Gáspár, Róbert

2016-01-01

Purpose Transdermal electroporation has become one of the most promising noninvasive methods for drug administration, with greatly increased transport of macromolecules through the skin. The cecal-contracting effects of repeated transdermal electroporation delivery and intravenous administration of neostigmine were compared in anesthetized rats. Methods The cecal contractions were detected with implantable strain gauge sensors, and the plasma levels of neostigmine were followed by high-performance liquid chromatography. Results Both intravenously and EP-administered neostigmine (0.2–66.7 μg/kg) increased the cecal contractions in a dose-dependent manner. For both the low doses and the highest dose, the neostigmine plasma concentrations were the same after the two modes of administration, while an insignificantly higher level was observed at a dose of 20 μg/kg after intravenous administration as compared with the electroporation route. The contractile responses did not differ significantly after the two administration routes. Conclusion The results suggest that electroporation-delivered neostigmine elicits action equivalent to that observed after intravenous administration as concerning both time and intensity. Electroporation permits the delivery of even lower doses of water-soluble compounds through the skin, which is very promising for clinical practice. PMID:27274203

A nonviral transfection approach in vitro: the design of a gold nanoparticle vector joint with microelectromechanical systems.

PubMed

Jen, Chun-Ping; Chen, Yu-Hung; Fan, Chun-sheng; Yeh, Chen-Sheng; Lin, Yu-Cheng; Shieh, Dar-Bin; Wu, Chao-Ling; Chen, Dong-Hwang; Chou, Chen-Hsi

2004-02-17

Au nanoparticles modified with 21-base thiolated-oligonucleotides have been evaluated as delivery vehicles for the development of a nonviral transfection platform. The electromigration combined with electroporation for DNA delivery in an osteoblast like cell was employed to test on microchips. Electroporation introduces foreign materials into cells by applying impulses of electric field to induce multiple transient pores on the cell membrane through dielectric breakdown of the cell membrane. On the basis of the characteristic surface plasmon of the Au particles, UV-vis absorption was utilized to qualitatively judge the efficiency of delivery. Transmission electron microscopy images and atomic absorption measurements (quantitative analysis) provided evidence of the bare Au and Au/oligonucleotide nanoparticles before and after electroporation and electromigration function. The experiments demonstrated that electrophoretic migration followed by electroporation significantly enhanced the transportation efficiency of the nanoparticle-oligonucleotide complexes as compared with electroporation alone. Most interestingly, Au capped with oligonucleotides led to optimal performance. On the other hand, the bare Au colloidal suspensions resulted in aggregation, which might be an obstacle to the internalization process. In addition, analytical results demonstrated an increase in the local particle concentrations on the cell surface that provided additional support for the mechanism underlying the improved Au nanoparticle transportation into cells in the presence of electromigration function.

Modeling of electric field distribution in tissues during electroporation

PubMed Central

2013-01-01

Background 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. Methods 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. Results 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 electric field distribution modeling in linear model of composite tissue (i.e. in the subcutaneous tumor model that do not take into account the relationship σ(E)) showed that a very high electric field (above irreversible threshold value) was concentrated only in the stratum corneum while the target tumor tissue was not successfully treated. Furthermore, the calculated volume of the target tumor tissue exposed to the electric field above reversible threshold in the subcutaneous model was zero assuming constant conductivities of each tissue. Our results also show that the inverse analysis allows for identification of both baseline tissue conductivity (i.e. conductivity of non-electroporated tissue) and tissue conductivity vs. electric field (σ(E)) of electroporated tissue. Conclusion Our results of modeling of electric field distribution in tissues during electroporation show that the changes in electrical conductivity due to electroporation need to be taken into account when an electroporation based treatment is planned or investigated. We concluded that the model of electric field distribution that takes into account the increase in electric conductivity due to electroporation yields more precise prediction of successfully electroporated target tissue volume. The findings of our study can significantly contribute to the current development of individualized patient-specific electroporation based treatment planning. PMID:23433433

High frequency electroporation efficiency is under control of membrane capacitive charging and voltage potential relaxation.

PubMed

Novickij, Vitalij; Ruzgys, Paulius; Grainys, Audrius; Šatkauskas, Saulius

2018-02-01

The study presents the proof of concept for a possibility to achieve a better electroporation in the MHz pulse repetition frequency (PRF) region compared to the conventional low frequency protocols. The 200ns×10 pulses bursts of 10-14kV/cm have been used to permeabilize Chinese hamster ovary (CHO) cells in a wide range (1Hz-1MHz) of PRF. The permeabilization efficiency was evaluated using fluorescent dye assay (propidium iodide) and flow cytometry. It was determined that a threshold PRF exists when the relaxation of the cell transmembrane potential is longer than the delay between the consequent pulses, which results in accumulation of the charge on the membrane. For the CHO cells and 0.1S/m electroporation medium, this phenomenon is detectable in the 0.5-1MHz range. It was shown that the PRF is an important parameter that could be used for flexible control of electroporation efficiency in the high frequency range. Copyright © 2017 Elsevier B.V. All rights reserved.

Electroporation-Induced Cell Modifications Detected with THz Time-Domain Spectroscopy

NASA Astrophysics Data System (ADS)

Romeo, Stefania; Vernier, P. Thomas; Zeni, Olga

2018-04-01

Electroporation (electropermeabilization) increases the electrical conductivity of biological cell membranes and lowers transport barriers for normally impermeant materials. Molecular simulations suggest that electroporation begins with the reorganization of water and lipid head group dipoles in the phospholipid bilayer interface, driven by an externally applied electric field, and the evolution of the resulting defects into water-filled, lipid pores. The interior of the electroporated membrane thus contains water, which should provide a signature for detection of the electropermeabilized state. In this feasibility study, we use THz time-domain spectroscopy, a powerful tool for investigating biomolecular systems and their interactions with water, to detect electroporation in human cells subjected to permeabilizing pulsed electric fields (PEFs). The time-domain response of electroporated human monocytes was acquired with a commercial THz, time-domain spectrometer. For each sample, frequency spectra were calculated, and the absorption coefficient and refractive index were extracted in the frequency range between 0.2 and 1.5 THz. This analysis reveals a higher absorption of THz radiation by PEF-exposed cells, with respect to sham-exposed ones, consistent with the intrusion of water into the cell through the permeabilized membrane that is presumed to be associated with electroporation.

Synergistic Combination of Electrolysis and Electroporation for Tissue Ablation.

PubMed

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.

Focal macromolecule delivery in neuronal tissue using simultaneous pressure ejection and local electroporation

PubMed Central

Barker, Matthew; Billups, Brian; Hamann, Martine

2009-01-01

Electroporation creates transient pores in the plasma membrane to introduce macromolecules within a cell or cell population. Generally, electrical pulses are delivered between two electrodes separated from each other, making electroporation less likely to be localised. We have developed a new device combining local pressure ejection with local electroporation through a double-barrelled glass micropipette to transfer impermeable macromolecules in brain slices or in cultured HEK293 cells. The design achieves better targeting of the site of pressure ejection with that of electroporation. With this technique, we have been able to limit the delivery of propidium iodide or dextran amine within areas of 100–200 μm diameter. We confirm that local electroporation is transient and show that when combined with pressure ejection, it allows local transfection of EGFP plasmids within HEK293 cells or within cerebellar and hippocampal slice cultures. We further show that local electroporation is less damaging when compared to global electroporation using two separate electrodes. Focal delivery of dextran amine dyes within trapezoid body fibres allowed tracing axonal tracts within brainstem slices, enabling the study of identified calyx of Held presynaptic terminals in living brain tissue. This labelling method can be used to target small nuclei in neuronal tissue and is generally applicable to the study of functional synaptic connectivity, or live axonal tracing in a variety of brain areas. PMID:19014970

A Numerical Investigation of the Electric and Thermal Cell Kill Distributions in Electroporation-Based Therapies in Tissue

PubMed Central

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 surrounding healthy tissue, critical vascular structures, and/or adjacent organs. PMID:25115970

Automated electrotransformation of Escherichia coli on a digital microfluidic platform using bioactivated magnetic beads.

PubMed

Moore, J A; Nemat-Gorgani, M; Madison, A C; Sandahl, M A; Punnamaraju, S; Eckhardt, A E; Pollack, M G; Vigneault, F; Church, G M; Fair, R B; Horowitz, M A; Griffin, P B

2017-01-01

This paper reports on the use of a digital microfluidic platform to perform multiplex automated genetic engineering (MAGE) cycles on droplets containing Escherichia coli cells. Bioactivated magnetic beads were employed for cell binding, washing, and media exchange in the preparation of electrocompetent cells in the electrowetting-on-dieletric (EWoD) platform. On-cartridge electroporation was used to deliver oligonucleotides into the cells. In addition to the optimization of a magnetic bead-based benchtop protocol for generating and transforming electrocompetent E. coli cells, we report on the implementation of this protocol in a fully automated digital microfluidic platform. Bead-based media exchange and electroporation pulse conditions were optimized on benchtop for transformation frequency to provide initial parameters for microfluidic device trials. Benchtop experiments comparing electrotransformation of free and bead-bound cells are presented. Our results suggest that dielectric shielding intrinsic to bead-bound cells significantly reduces electroporation field exposure efficiency. However, high transformation frequency can be maintained in the presence of magnetic beads through the application of more intense electroporation pulses. As a proof of concept, MAGE cycles were successfully performed on a commercial EWoD cartridge using variations of the optimal magnetic bead-based preparation procedure and pulse conditions determined by the benchtop results. Transformation frequencies up to 22% were achieved on benchtop; this frequency was matched within 1% (21%) by MAGE cycles on the microfluidic device. However, typical frequencies on the device remain lower, averaging 9% with a standard deviation of 9%. The presented results demonstrate the potential of digital microfluidics to perform complex and automated genetic engineering protocols.

Automated electrotransformation of Escherichia coli on a digital microfluidic platform using bioactivated magnetic beads

PubMed Central

Moore, J. A.; Nemat-Gorgani, M.; Madison, A. C.; Punnamaraju, S.; Eckhardt, A. E.; Pollack, M. G.; Church, G. M.; Fair, R. B.; Horowitz, M. A.; Griffin, P. B.

2017-01-01

This paper reports on the use of a digital microfluidic platform to perform multiplex automated genetic engineering (MAGE) cycles on droplets containing Escherichia coli cells. Bioactivated magnetic beads were employed for cell binding, washing, and media exchange in the preparation of electrocompetent cells in the electrowetting-on-dieletric (EWoD) platform. On-cartridge electroporation was used to deliver oligonucleotides into the cells. In addition to the optimization of a magnetic bead-based benchtop protocol for generating and transforming electrocompetent E. coli cells, we report on the implementation of this protocol in a fully automated digital microfluidic platform. Bead-based media exchange and electroporation pulse conditions were optimized on benchtop for transformation frequency to provide initial parameters for microfluidic device trials. Benchtop experiments comparing electrotransformation of free and bead-bound cells are presented. Our results suggest that dielectric shielding intrinsic to bead-bound cells significantly reduces electroporation field exposure efficiency. However, high transformation frequency can be maintained in the presence of magnetic beads through the application of more intense electroporation pulses. As a proof of concept, MAGE cycles were successfully performed on a commercial EWoD cartridge using variations of the optimal magnetic bead-based preparation procedure and pulse conditions determined by the benchtop results. Transformation frequencies up to 22% were achieved on benchtop; this frequency was matched within 1% (21%) by MAGE cycles on the microfluidic device. However, typical frequencies on the device remain lower, averaging 9% with a standard deviation of 9%. The presented results demonstrate the potential of digital microfluidics to perform complex and automated genetic engineering protocols. PMID:28191268

Avoiding nerve stimulation in irreversible electroporation: a numerical modeling study

NASA Astrophysics Data System (ADS)

Mercadal, Borja; Arena, Christopher B.; Davalos, Rafael V.; Ivorra, Antoni

2017-10-01

Electroporation based treatments consist in applying one or multiple high voltage pulses to the tissues to be treated. As an undesired side effect, these pulses cause electrical stimulation of excitable tissues such as nerves and muscles. This increases the complexity of the treatments and may pose a risk to the patient. To minimize electrical stimulation during electroporation based treatments, it has been proposed to replace the commonly used monopolar pulses by bursts of short bipolar pulses. In the present study, we have numerically analyzed the rationale for such approach. We have compared different pulsing protocols in terms of their electroporation efficacy and their capability of triggering action potentials in nerves. For that, we have developed a modeling framework that combines numerical models of nerve fibers and experimental data on irreversible electroporation. Our results indicate that, by replacing the conventional relatively long monopolar pulses by bursts of short bipolar pulses, it is possible to ablate a large tissue region without triggering action potentials in a nearby nerve. Our models indicate that this is possible because, as the pulse length of these bipolar pulses is reduced, the stimulation thresholds raise faster than the irreversible electroporation thresholds. We propose that this different dependence on the pulse length is due to the fact that transmembrane charging for nerve fibers is much slower than that of cells treated by electroporation because of their geometrical differences.

Mutated-leptin gene transfer induces increases in body weight by electroporation and hydrodynamics-based gene delivery in mice.

PubMed

Xiang, Lan; Murai, Atsushi; Muramatsu, Tatsuo

2005-12-01

To investigate whether in vivo gene transfer causes leptin-antagonistic effects on food intake, animal body weight and fat tissue weight, the R128Q mutated-leptin gene, an R to Q substitution at position 128 of mouse leptin, was transferred into mouse liver and leg muscle by electroporation and hydrodynamics-based gene delivery. Mutated-leptin gene transfer by electroporation caused significant increases in body weight at 5 days and after (5.4% increase relative to control; p<0.05). Hydrodynamics-based gene delivery of the mutated-leptin gene also caused an increase in body weight (3.0% increase relative to control; p<0.05). Mutated-leptin gene transfer by electroporation significantly increased the tissue weight of epididymal white fat and neuropeptide Y mRNA expression in the hypothalamus compared with those of the control group 3 weeks after gene transfer (p<0.05). These results suggest that mutated-leptin gene transfer successfully produced leptin-antagonistic effects by modulating the central regulator of energy homeostasis. Also, the extent of leptin-antagonistic effects by electroporation was much higher than hydrodynamics-based gene delivery, with at least single gene transfer.

Cell Electrosensitization Exists Only in Certain Electroporation Buffers.

PubMed

Dermol, Janja; Pakhomova, Olga N; Pakhomov, Andrei G; Miklavčič, Damijan

2016-01-01

Electroporation-induced cell sensitization was described as the occurrence of a delayed hypersensitivity to electric pulses caused by pretreating cells with electric pulses. It was achieved by increasing the duration of the electroporation treatment at the same cumulative energy input. It could be exploited in electroporation-based treatments such as electrochemotherapy and tissue ablation with irreversible electroporation. The mechanisms responsible for cell sensitization, however, have not yet been identified. We investigated cell sensitization dynamics in five different electroporation buffers. We split a pulse train into two trains varying the delay between them and measured the propidium uptake by fluorescence microscopy. By fitting the first-order model to the experimental results, we determined the uptake due to each train (i.e. the first and the second) and the corresponding resealing constant. Cell sensitization was observed in the growth medium but not in other tested buffers. The effect of pulse repetition frequency, cell size change, cytoskeleton disruption and calcium influx do not adequately explain cell sensitization. Based on our results, we can conclude that cell sensitization is a sum of several processes and is buffer dependent. Further research is needed to determine its generality and to identify underlying mechanisms.

Cell Electrosensitization Exists Only in Certain Electroporation Buffers

PubMed Central

Dermol, Janja; Pakhomova, Olga N.; Pakhomov, Andrei G.; Miklavčič, Damijan

2016-01-01

Electroporation-induced cell sensitization was described as the occurrence of a delayed hypersensitivity to electric pulses caused by pretreating cells with electric pulses. It was achieved by increasing the duration of the electroporation treatment at the same cumulative energy input. It could be exploited in electroporation-based treatments such as electrochemotherapy and tissue ablation with irreversible electroporation. The mechanisms responsible for cell sensitization, however, have not yet been identified. We investigated cell sensitization dynamics in five different electroporation buffers. We split a pulse train into two trains varying the delay between them and measured the propidium uptake by fluorescence microscopy. By fitting the first-order model to the experimental results, we determined the uptake due to each train (i.e. the first and the second) and the corresponding resealing constant. Cell sensitization was observed in the growth medium but not in other tested buffers. The effect of pulse repetition frequency, cell size change, cytoskeleton disruption and calcium influx do not adequately explain cell sensitization. Based on our results, we can conclude that cell sensitization is a sum of several processes and is buffer dependent. Further research is needed to determine its generality and to identify underlying mechanisms. PMID:27454174

Synergistic Combination of Electrolysis and Electroporation for Tissue Ablation

PubMed Central

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. PMID:26866693

Irreversible electroporation of stage 3 locally advanced pancreatic cancer: optimal technique and outcomes

PubMed Central

2015-01-01

Objective Irreversible electroporation (IRE) of stage 3 pancreatic adenocarcinoma has been used to provide quality of life time in patients who have undergone appropriate induction therapy. The optimal technique has been reported within the literature, but not in video form. IRE of locally advanced pancreatic cancer is technically demanding requiring precision ultrasound use for continuous imaging in multiple needle placements and during IRE energy delivery. Methods Appropriate patients with locally advanced pancreatic cancer should have undergone appropriate induction chemotherapy for a reasonable duration. The safe and effective technique for irreversible electroporation is preformed through an open approach with the emphasis on intra-operative ultrasound and intra-operative electroporation management. Results The technique of open irreversible electroporation of the pancreas involves bracketing the target tumor with IRE probes and any and all invaded vital structures including the celiac axis, superior mesenteric artery (SMA), superior mesenteric-portal vein, and bile duct with continuous intraoperative ultrasound imaging through a caudal to cranial approach. Optimal IRE delivery requires a change in amperage of at least 12 amps from baseline tissue conductivity in order to achieve technical success. Multiple pull-backs are necessary since the IRE ablation probe lengths are 1 cm and thus needed to achieve technical success along the caudal to cranial plane. Conclusions Irreversible electroporation in combination with multi-modality therapy for locally advanced pancreatic carcinoma is feasible for appropriate patients with locally advanced cancer. Technical demands are high and require the highest quality ultrasound for precise spacing measurements and optimal delivery to ensure adequate change in tissue resistance. PMID:29075594

The effect of temperature and bacterial growth phase on protein extraction by means of electroporation.

PubMed

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Planning of electroporation-based treatments using Web-based treatment-planning software.

PubMed

Pavliha, Denis; Kos, Bor; Marčan, Marija; Zupanič, Anže; Serša, Gregor; Miklavčič, Damijan

2013-11-01

Electroporation-based treatment combining high-voltage electric pulses and poorly permanent cytotoxic drugs, i.e., electrochemotherapy (ECT), is currently used for treating superficial tumor nodules by following standard operating procedures. Besides ECT, another electroporation-based treatment, nonthermal irreversible electroporation (N-TIRE), is also efficient at ablating deep-seated tumors. To perform ECT or N-TIRE of deep-seated tumors, following standard operating procedures is not sufficient and patient-specific treatment planning is required for successful treatment. Treatment planning is required because of the use of individual long-needle electrodes and the diverse shape, size and location of deep-seated tumors. Many institutions that already perform ECT of superficial metastases could benefit from treatment-planning software that would enable the preparation of patient-specific treatment plans. To this end, we have developed a Web-based treatment-planning software for planning electroporation-based treatments that does not require prior engineering knowledge from the user (e.g., the clinician). The software includes algorithms for automatic tissue segmentation and, after segmentation, generation of a 3D model of the tissue. The procedure allows the user to define how the electrodes will be inserted. Finally, electric field distribution is computed, the position of electrodes and the voltage to be applied are optimized using the 3D model and a downloadable treatment plan is made available to the user.

Maximizing Exosome Colloidal Stability Following Electroporation

PubMed Central

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

2014-01-01

Development of exosome based semi-synthetic 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 5 nm 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 to enrich exogenously modified exosomes and introduces the potential for MRI driven theranostic exosome investigations in vivo. PMID:24333249

Maximizing exosome colloidal stability following electroporation.

PubMed

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. Copyright © 2013 Elsevier Inc. All rights reserved.

Administration of HPV DNA vaccine via electroporation elicits the strongest CD8+ T cell immune responses compared to intramuscular injection and intradermal gene gun delivery

PubMed Central

Best, Simon R.; Peng, Shiwen; Juang, Chi-Mou; Hung, Chien-Fu; Hannaman, Drew; Saunders, John R.; Wu, T.-C.; Pai, Sara I.

2009-01-01

DNA vaccines are an attractive approach to eliciting antigen-specific immunity. Intracellular targeting of tumor antigens through its linkage to immunostimulatory molecules such as calreticulin (CRT) can improve antigen processing and presentation through the MHC Class I pathway and increase cytotoxic CD8+ T cell production. However, even with these enhancements, the efficacy of such immunotherapeutic strategies is dependent on the identification of an effective route and method of DNA administration. Electroporation and gene gun-mediated particle delivery are leading methods of DNA vaccine delivery that can generate protective and therapeutic levels of immune responses in experimental models. In this study, we perform a head-to-head comparison of three methods of vaccination – conventional intramuscular injection, electroporation mediated intramuscular delivery, and epidermal gene gun-mediated particle delivery - in the ability to generate antigen specific cytotoxic CD8+ T cell responses as well as anti-tumor immune responses against an HPV-16 E7 expressing tumor cell line using the pNGVL4a-CRT/E7(detox) DNA vaccine. Vaccination via electroporation generated the highest number of E7-specific cytotoxic CD8+ T cells, which correlated to improved outcomes in the treatment of growing tumors. In addition, we demonstrate that electroporation results in significantly higher levels of circulating protein compared to gene gun or intramuscular vaccination, which likely enhances calreticulin’s role as a local tumor anti-angiogenesis agent. We conclude that electroporation is a promising method for delivery of HPV DNA vaccines and should be considered for DNA vaccine delivery in human clinical trials. PMID:19622402

Transient oscillation of shape and membrane conductivity changes by field pulse-induced electroporation in nano-sized phospholipid vesicles.

PubMed

Dimitrov, Vasil; Kakorin, Sergej; Neumann, Eberhard

2013-05-07

The results of electrooptical and conductometrical measurements on unilamellar lipid vesicles (of mean radius a = 90 nm), filled with 0.2 M NaCl solution, suspended in 0.33 M sucrose solution of 0.2 mM NaCl, and exposed to a stepwise decaying electric field (time constant τE = 154 μs) in the range 10 ≤ E0 (kV cm(-1)) ≤ 90, are analyzed in terms of cyclic changes in vesicle shape and vesicle membrane conductivity. The two peaks in the dichroitic turbidity relaxations reflect two cycles of rapid membrane electroporation and slower resealing of long-lived electropores. The field-induced changes reflect structural transitions between closed (C) and porated (P) membrane states, qualified by pores of type P1 and of type P2, respectively. The transient change in the membrane conductivity and the transient shape oscillation are based on changes in the pore density of the (larger) P2-pores along a hysteresis cycle. The P2-pore formation leads to transient net ion flows across the P2-pores and to transient changes in the membrane field. The kinetic data are numerically processed in terms of coupled structural relaxation modes. Using the torus-hole pore model, the mean inner pore radii are estimated to be r1 = 0.38 (±0.05) nm and r2 = 1.7 (±0.1) nm, respectively. The observation of a transient oscillation of membrane electroporation and of shape changes in a longer lasting external field pulse is suggestive of potential resonance enhancement, for instance, of electro-uptake by, and of electro-release of biogenic molecules from, biological cells in trains of long-lasting low-intensity voltage pulses.

A statistical model describing combined irreversible electroporation and electroporation-induced blood-brain barrier disruption.

PubMed

Sharabi, Shirley; Kos, Bor; Last, David; Guez, David; Daniels, Dianne; Harnof, Sagi; Mardor, Yael; Miklavcic, Damijan

2016-03-01

Electroporation-based therapies such as electrochemotherapy (ECT) and irreversible electroporation (IRE) are emerging as promising tools for treatment of tumors. When applied to the brain, electroporation can also induce transient blood-brain-barrier (BBB) disruption in volumes extending beyond IRE, thus enabling efficient drug penetration. The main objective of this study was to develop a statistical model predicting cell death and BBB disruption induced by electroporation. This model can be used for individual treatment planning. Cell death and BBB disruption models were developed based on the Peleg-Fermi model in combination with numerical models of the electric field. The model calculates the electric field thresholds for cell kill and BBB disruption and describes the dependence on the number of treatment pulses. The model was validated using in vivo experimental data consisting of rats brains MRIs post electroporation treatments. Linear regression analysis confirmed that the model described the IRE and BBB disruption volumes as a function of treatment pulses number (r(2) = 0.79; p < 0.008, r(2) = 0.91; p < 0.001). The results presented a strong plateau effect as the pulse number increased. The ratio between complete cell death and no cell death thresholds was relatively narrow (between 0.88-0.91) even for small numbers of pulses and depended weakly on the number of pulses. For BBB disruption, the ratio increased with the number of pulses. BBB disruption radii were on average 67% ± 11% larger than IRE volumes. The statistical model can be used to describe the dependence of treatment-effects on the number of pulses independent of the experimental setup.

Changes in optical properties of electroporated cells as revealed by digital holographic microscopy

PubMed Central

Calin, Violeta L.; Mihailescu, Mona; Mihale, Nicolae; Baluta, Alexandra V.; Kovacs, Eugenia; Savopol, Tudor; Moisescu, Mihaela G.

2017-01-01

Changes in optical and shape-related characteristics of B16F10 cells after electroporation were investigated using digital holographic microscopy (DHM). Bipolar rectangular pulses specific for electrochemotherapy were used. Electroporation was performed in an “off-axis” DHM set-up without using exogenous markers. Two types of cell parameters were monitored seconds and minutes after pulse train application: parameters addressing a specifically defined area of the cell (refractive index and cell height) and global cell parameters (projected area, optical phase shift profile and dry mass). The biphasic behavior of cellular parameters was explained by water and mannitol dynamics through the electropermeabilized cell membrane. PMID:28736667

Modeling in conventional and supra electroporation for model cell with organelles

NASA Astrophysics Data System (ADS)

Sulaeman, Muhammad Yangki; Widita, Rena

2015-09-01

Electroporation is a formation of pores in the membrane cell due to the external electric field applied to the cell. There are two types of electroporation, conventional and supra-electroporation. The purpose of creating pores in the cell using conventional electroporation are to increase the effectiveness of chemotherapy (electrochemotherapy) and to kill cancer tissue using irreversible electroporation. Supra-electroporation shows that it can induce electroporation in the organell inside the cell, so it can kill the cell by apoptosis mechanism. Modeling of electroporation phenomenon on a model cell had been done by using software COMSOL Multiphysics 4.3b with the applied external electric field used are 1.1 kV/cm for conventional electroporation and 60 kV/cm for supra-electroporation to find the difference between transmembrane voltage and pore density for both electroporation. It can be concluded from the results that there is a big difference between transmembrane voltage and pores density on conventional and supra electroporation on model cell.

Bleomycin--electrical pulse delivery: electroporation therapy-bleomycin--Genetronics; MedPulser-bleomycin--Genetronics.

PubMed

2004-01-01

Genetronics Biomedical is using its electroporation therapy technology to deliver bleomycin to tumour cells for the treatment of cancer. Genetronics have developed the MedPulser Electroporation Therapy System, which consists of an electrical pulse generator and disposable electrode applicators. The MedPulser system enables the delivery of large molecules into cells by briefly applying an electric field to the cell. This causes a transient permeability in the cell's outer membrane characterised by the appearance of pores across the membrane. After the field is discontinued, the pores close, trapping the therapeutic molecules inside the target cells. Genetronics is using the MedPulser System in conjunction with bleomycin, an antineoplastic antibiotic that binds to DNA causing strand scissions. Genetronics is seeking a licensing partner for the use of electroporation for the delivery of drugs in chemotherapy. In 1998, Genetronics entered a licensing and development agreement with Ethicon for electroporation and electrofusion. Under the terms of this agreement, Ethicon was to develop and clinically test the Genetronics electroporation delivery system and conduct all regulatory activities throughout the world except Canada. Ethicon would also market the products once regulatory approval has been obtained and Genetronics was to receive a percentage of the net sales and as license fees. However, in July 2000, Ethicon exercised its rights to terminate the agreement without cause. All rights were returned to Genetronics in January 2001. In 1997, Genetronics entered an agreement with Abbott Laboratories for the manufacture of bleomycin for use in the US in its MedPulsar system after regulatory approval had been granted for its use in the treatment of solid tumours. In a separate supply agreement, Faulding Inc. has agreed to manufacture bleomycin for Genetronic for use in Canada after regulatory approval had been granted. The MedPulsar Electroporation Therapy System with bleomycin is currently in phase III pivotal studies in the US as a treatment for recurrent and second primary squamous cell carcinomas of the head and neck. Genetronics received approval for the Electroporation Therapy system as a device in March 1999 when it achieved CE Mark certification. In February 2004, Genetronics announced that it had completed a Special Protocol Assessment review process with the US FDA for two new trials that will compare bleomycin electroporation therapy to surgery. The primary endpoint will be tissue and function preservation rather than survival. One proposal is for recurrent head and neck cancer, and the other is for disfiguring cutaneous cancer. Three Institutional Review Boards in the US have approved the two protocols and Genetronics has initiated enrollment. In June 2004, Genetronics was granted fast-track status for its MedPulsar Electroporation Therapy System clinical development programme for patients with head and neck cancer. Shifting from a primary endpoint of survival to a quality-of-life outcome will enable those clinical trials to be carried out faster with less cost and with a higher likelihood of success. As a result, Genetronic's phase III trials focussing on survival as a primary endpoint have been discontinued. This includes a phase III trial for late-stage, recurrent head and neck cancer in combination with the normal standard of treatment compared with normal standard of treatment alone. Interim results from this trial had suggested bleomycin electroporation therapy demonstrated local tumour control and preservation of organ function, as well as non-inferiority when compared with surgery. This trial was initiated in May 2002. In March 2004, Genetronics initiated a post-European regulatory approval clinical study in patients with primary or recurrent squamous cell carcinoma of the head and neck (SCCHN). This study aims to enroll approximately 100 patients at 12-15 hospitals located in the UK, Germany, Italy, France, Austria and other western European countries. The study is designed to support the commercialisation of the MedPulser Electroporation System in the EU. Prior clinical trials established the safety and performance of the MedPulser System for the treatment of SCCHN, leading to approval for sale in the EU based on achieving the CE Mark. This study will document the clinical and pharmacoeconomic benefit in support of reimbursement approval throughout Western Europe, establish centres of excellence to facilitate early sales, create a reference and customer base for a projected European commercial launch in 2005, and generate safety and efficacy data to support marketing applications in the US. The bleomycin delivery system has completed phase IIB trials in the US, Canada and Europe in patients with squamous cell carcinoma of the head and neck who have failed conventional therapies. Phase II data were submitted to the FDA in the first quarter of 2002 and a phase III trial was launched in May 2002. The therapy is also being used in France in patients with cancers of the head and neck, liver (metastatic) and melanoma. A review of the data from these phase II trials was completed in April 2001. In June 2004, Genetronics was granted two US patents. US patent 6,748,265 covers its trans-surface drug and gene delivery technology and provides additional proprietary rights for an apparatus and method to deliver genes, drugs and other molecules through tissue surfaces. The second US patent, 6,746,441, pertains to the field of ex vivo therapies and covers the introduction of molecules into cells by electroporation, either in a continuous-flow or batch mode, with a variable electric field orientation. In July 2004, Genetronics received a US patent (no. 6,763,264) covering methods for the in vivo delivery of a recombinant expression vector (DNA) or a pharmaceutical agent into tissue cells, and a method for the therapeutic application of electroporation to a patient to introduce macromolecules. Copyright 2004 Adis Data Information BV

A statistical model describing combined irreversible electroporation and electroporation-induced blood-brain barrier disruption

PubMed Central

Sharabi, Shirley; Kos, Bor; Last, David; Guez, David; Daniels, Dianne; Harnof, Sagi; Miklavcic, Damijan

2016-01-01

Background Electroporation-based therapies such as electrochemotherapy (ECT) and irreversible electroporation (IRE) are emerging as promising tools for treatment of tumors. When applied to the brain, electroporation can also induce transient blood-brain-barrier (BBB) disruption in volumes extending beyond IRE, thus enabling efficient drug penetration. The main objective of this study was to develop a statistical model predicting cell death and BBB disruption induced by electroporation. This model can be used for individual treatment planning. Material and methods Cell death and BBB disruption models were developed based on the Peleg-Fermi model in combination with numerical models of the electric field. The model calculates the electric field thresholds for cell kill and BBB disruption and describes the dependence on the number of treatment pulses. The model was validated using in vivo experimental data consisting of rats brains MRIs post electroporation treatments. Results Linear regression analysis confirmed that the model described the IRE and BBB disruption volumes as a function of treatment pulses number (r2 = 0.79; p < 0.008, r2 = 0.91; p < 0.001). The results presented a strong plateau effect as the pulse number increased. The ratio between complete cell death and no cell death thresholds was relatively narrow (between 0.88-0.91) even for small numbers of pulses and depended weakly on the number of pulses. For BBB disruption, the ratio increased with the number of pulses. BBB disruption radii were on average 67% ± 11% larger than IRE volumes. Conclusions The statistical model can be used to describe the dependence of treatment-effects on the number of pulses independent of the experimental setup. PMID:27069447

Irreversible electroporation in the treatment of locally advanced pancreas and liver metastases of colorectal carcinoma.

PubMed

Wichtowski, Mateusz; Nowaczyk, Piotr; Kocur, Jacek; Murawa, Dawid

2016-01-01

Irreversible electroporation is a new, non-thermal ablation technique in the treatment of parenchymal organ tumors which uses short high voltage pulses of electricity in order to induce apoptosis of targeted cells. In this paper the application of this method of treatment in locally advanced pancreatic cancer (LAPC) and liver cancer is analyzed. Between 04.2014 and 09.2014 two patients with LAPC and one with colorectal liver metastasis (CRLM) were qualified for treatment with irreversible electroporation. Both patients remained under constant observation and control. PubMed/Medline, Embase and Google Scholar databases were searched and eight original reports on irreversible electroporation of pancreatic and liver tumors based on the biggest groups of patients were found. Two patients with LAPC and one with CRLM were qualified for ablation with irreversible electroporation. In all three patients a successful irreversible electroporation (IRE) procedure of the whole tumor was conducted. In the minimum seven-month follow-up 100% local control was achieved - without progression. In the literature review the local response to treatment ranged from 41% to 100%. The event-free survival rate in six-month observation was 94%. Ablation with irreversible electroporation is a new non-thermal ablation technique which has been demonstrated, both in the previously published studies and in the cases described in this paper, as a safe and efficient therapeutic method for patients with LAPC and CRLM.

Transfection of isolated rainbow trout, Oncorhynchus mykiss, granulosa cells through chemical transfection and electroporation at 12°C.

PubMed

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. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparative performance of a licensed anthrax vaccine versus electroporation based delivery of a PA encoding DNA vaccine in rhesus macaques.

PubMed

Livingston, Brian D; Little, Stephen F; Luxembourg, Alain; Ellefsen, Barry; Hannaman, Drew

2010-01-22

DNA vaccination is a promising immunization strategy that could be applied in the development of vaccines for a variety of prophylactic and therapeutic indications. Utilizing anthrax protective antigen as a model antigen, we demonstrate that electroporation mediated delivery enhanced the immunogenicity of DNA vaccines in nonhuman primates over 100-fold as compared to conventional intramuscular injection. Two administrations of a DNA vaccine with electroporation elicited anthrax toxin neutralizing antibody responses in 100% of rhesus macaques. Toxin neutralizing antibodies were sustained for the nearly 1-year study duration and were correlated with protection against subsequent lethal Bacillus anthracis spore challenge. Collectively, electroporation mediated DNA vaccination conferred protection comparable to that observed following vaccination with an FDA approved anthrax vaccine.

A molecular insight into the electro-transfer of small molecules through electropores driven by electric fields.

PubMed

Casciola, Maura; Tarek, Mounir

2016-10-01

The transport of chemical compounds across the plasma membrane into the cell is relevant for several biological and medical applications. One of the most efficient techniques to enhance this uptake is reversible electroporation. Nevertheless, the detailed molecular mechanism of transport of chemical species (dyes, drugs, genetic materials, …) following the application of electric pulses is not yet fully elucidated. In the past decade, molecular dynamics (MD) simulations have been conducted to model the effect of pulsed electric fields on membranes, describing several aspects of this phenomenon. Here, we first present a comprehensive review of the results obtained so far modeling the electroporation of lipid membranes, then we extend these findings to study the electrotransfer across lipid bilayers subject to microsecond pulsed electric fields of Tat11, a small hydrophilic charged peptide, and of siRNA. We use in particular a MD simulation protocol that allows to characterize the transport of charged species through stable pores. Unexpectedly, our results show that for an electroporated bilayer subject to transmembrane voltages in the order of 500mV, i.e. consistent with experimental conditions, both Tat11 and siRNA can translocate through nanoelectropores within tens of ns. We discuss these results in comparison to experiments in order to rationalize the mechanism of drug uptake by cells. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. Copyright © 2016 Elsevier B.V. All rights reserved.

Irreversible electroporation in the treatment of locally advanced pancreas and liver metastases of colorectal carcinoma

PubMed Central

Wichtowski, Mateusz; Nowaczyk, Piotr; Kocur, Jacek

2016-01-01

Aim of the study Irreversible electroporation is a new, non-thermal ablation technique in the treatment of parenchymal organ tumors which uses short high voltage pulses of electricity in order to induce apoptosis of targeted cells. In this paper the application of this method of treatment in locally advanced pancreatic cancer (LAPC) and liver cancer is analyzed. Material and methods Between 04.2014 and 09.2014 two patients with LAPC and one with colorectal liver metastasis (CRLM) were qualified for treatment with irreversible electroporation. Both patients remained under constant observation and control. PubMed/Medline, Embase and Google Scholar databases were searched and eight original reports on irreversible electroporation of pancreatic and liver tumors based on the biggest groups of patients were found. Results Two patients with LAPC and one with CRLM were qualified for ablation with irreversible electroporation. In all three patients a successful irreversible electroporation (IRE) procedure of the whole tumor was conducted. In the minimum seven-month follow-up 100% local control was achieved – without progression. In the literature review the local response to treatment ranged from 41% to 100%. The event-free survival rate in six-month observation was 94%. Conclusions Ablation with irreversible electroporation is a new non-thermal ablation technique which has been demonstrated, both in the previously published studies and in the cases described in this paper, as a safe and efficient therapeutic method for patients with LAPC and CRLM. PMID:27095938

Variation in dielectric properties due to pathological changes in human liver.

PubMed

Peyman, Azadeh; Kos, Bor; Djokić, Mihajlo; Trotovšek, Blaž; Limbaeck-Stokin, Clara; Serša, Gregor; Miklavčič, Damijan

2015-12-01

Dielectric properties of freshly excised human liver tissues (in vitro) with several pathological conditions including cancer were obtained in frequency range 100 MHz-5 GHz. Differences in dielectric behavior of normal and pathological tissues at microwave frequencies are discussed based on histological information for each tissue. Data presented are useful for many medical applications, in particular nanosecond pulsed electroporation techniques. Knowledge of dielectric properties is vital for mathematical calculations of local electric field distribution inside electroporated tissues and can be used to optimize the process of electroporation for treatment planning procedures. © 2015 Wiley Periodicals, Inc.

Nanochannel Electroporation as a Platform for Living Cell Interrogation in Acute Myeloid Leukemia.

PubMed

Zhao, Xi; Huang, Xiaomeng; Wang, Xinmei; Wu, Yun; Eisfeld, Ann-Kathrin; Schwind, Sebastian; Gallego-Perez, Daniel; Boukany, Pouyan E; Marcucci, Guido I; Lee, Ly James

2015-12-01

A living cell interrogation platform based on nanochannel electroporation is demonstrated with analysis of RNAs in single cells. This minimally invasive process is based on individual cells and allows both multi-target analysis and stimulus-response analysis by sequential deliveries. The unique platform possesses a great potential to the comprehensive and lysis-free nucleic acid analysis on rare or hard-to-transfect cells.

Electrical conductivity changes during irreversible electroporation treatment of brain cancer.

PubMed

Garcia, Paulo A; Rossmeisl, John H; Davalos, Rafael V

2011-01-01

Irreversible electroporation (IRE) is a new minimally invasive technique to kill tumors and other undesirable tissue in a non-thermal manner. During an IRE treatment, a series of short and intense electric pulses are delivered to the region of interest to destabilize the cell membranes in the tissue and achieve spontaneous cell death. The alteration of the cellular membrane results in a dramatic increase in electrical conductivity during IRE as in other electroporation-based-therapies. In this study, we performed the planning and execution of an IRE brain cancer treatment using MRI reconstructions of the tumor and a multichannel array that served as a stereotactic fiducial and electrode guide. Using the tumor reconstructions within our numerical simulations, we developed equations relating the increase in tumor conductivity to calculated currents and volumes of tumor treated with IRE. We also correlated the experimental current measured during the procedure to an increase in tumor conductivity ranging between 3.42-3.67 times the baseline conductivity, confirming the physical phenomenon that has been detected in other tissues undergoing similar electroporation-based treatments.

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

NASA Astrophysics Data System (ADS)

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.

Avoiding neuromuscular stimulation in liver irreversible electroporation using radiofrequency electric fields

NASA Astrophysics Data System (ADS)

Castellví, Quim; Mercadal, Borja; Moll, Xavier; Fondevila, Dolors; Andaluz, Anna; Ivorra, Antoni

2018-02-01

Electroporation-based treatments typically consist of the application of high-voltage dc pulses. As an undesired side effect, these dc pulses cause electrical stimulation of excitable tissues such as motor nerves. The present in vivo study explores the use of bursts of sinusoidal voltage in a frequency range from 50 kHz to 2 MHz, to induce irreversible electroporation (IRE) whilst avoiding neuromuscular stimulation. A series of 100 dc pulses or sinusoidal bursts, both with an individual duration of 100 µs, were delivered to rabbit liver through thin needles in a monopolar electrode configuration, and thoracic movements were recorded with an accelerometer. Tissue samples were harvested three hours after treatment and later post-processed to determine the dimensions of the IRE lesions. Thermal damage due to Joule heating was ruled out via computer simulations. Sinusoidal bursts with a frequency equal to or above 100 kHz did not cause thoracic movements and induced lesions equivalent to those obtained with conventional dc pulses when the applied voltage amplitude was sufficiently high. IRE efficacy dropped with increasing frequency. For 100 kHz bursts, it was estimated that the electric field threshold for IRE is about 1.4 kV cm-1 whereas that of dc pulses is about 0.5 kV cm-1.

Electroporation of Postimplantation Mouse Embryos In Utero.

PubMed

Huang, Cheng-Chiu; Carcagno, Abel

2018-02-01

Gene transfer by electroporation is possible in mouse fetuses within the uterus. As described in this protocol, the pregnant female is anesthetized, the abdominal cavity is opened, and the uterus with the fetuses is exteriorized. A solution of plasmid DNA is injected through the uterine wall directly into the fetus, typically into a cavity like the brain ventricle, guided by fiber optic illumination. Electrodes are positioned on the uterus around the region of the fetus that was injected, and electrical pulses are delivered. The uterus is returned to the abdominal cavity, the body wall is sutured closed, and the female is allowed to recover. The manipulated fetuses can then be collected and analyzed at various times after the electroporation. This method allows experimental access to later-stage developing mouse embryos. © 2018 Cold Spring Harbor Laboratory Press.

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

PubMed

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. © 2013 Elsevier B.V. All rights reserved.

An Infrared Actin Probe for Deep-Cell Electroporation-Based Single-Molecule Speckle (eSiMS) Microscopy

PubMed Central

Yamashiro, Sawako; Watanabe, Naoki

2017-01-01

Single-molecule speckle (SiMS) microscopy is a powerful method to directly elucidate biochemical reactions in live cells. However, since the signal from an individual fluorophore is extremely faint, the observation area by epi-fluorescence microscopy is restricted to the thin cell periphery to reduce autofluorescence, or only molecules near the plasma membrane are visualized by total internal reflection fluorescence (TIRF) microscopy. Here, we introduce a new actin probe labeled with near infrared (NIR) emissive CF680R dye for easy-to-use, electroporation-based SiMS microscopy (eSiMS) for deep-cell observation. CF680R-labeled actin (CF680R-actin) incorporated into actin structures and showed excellent brightness and photostability suitable for single-molecule imaging. Importantly, the intensity of autofluorescence with respect to SiMS brightness was reduced to approximately 13% compared to DyLight 550-labeled actin (DL550-actin). CF680R-actin enabled the monitoring of actin SiMS in actomyosin bundles associated with adherens junctions (AJs) located at 3.5–4 µm above the basal surfaces of epithelial monolayers. These favorable properties of CF680R-actin extend the application of eSiMS to actin turnover and flow analyses in deep cellular structures. PMID:28671584

Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique.

PubMed

Xiong, Bin; Li, Zhongkang; Liu, Li; Zhao, Dongdong; Zhang, Xueli; Bi, Changhao

2018-01-01

Ralstonia eutropha is an important bacterium for the study of polyhydroxyalkanoates (PHAs) synthesis and CO 2 fixation, which makes it a potential strain for industrial PHA production and attractive host for CO 2 conversion. Although the bacterium is not recalcitrant to genetic manipulation, current methods for genome editing based on group II introns or single crossover integration of a suicide plasmid are inefficient and time-consuming, which limits the genetic engineering of this organism. Thus, developing an efficient and convenient method for R. eutropha genome editing is imperative. An efficient genome editing method for R. eutropha was developed using an electroporation-based CRISPR-Cas9 technique. In our study, the electroporation efficiency of R. eutropha was found to be limited by its restriction-modification (RM) systems. By searching the putative RM systems in R. eutropha H16 using REBASE database and comparing with that in E. coli MG1655, five putative restriction endonuclease genes which are related to the RM systems in R. eutropha were predicated and disrupted. It was found that deletion of H16_A0006 and H16_A0008 - 9 increased the electroporation efficiency 1658 and 4 times, respectively. Fructose was found to reduce the leaky expression of the arabinose-inducible pBAD promoter, which was used to optimize the expression of cas9 , enabling genome editing via homologous recombination based on CRISPR-Cas9 in R. eutropha . A total of five genes were edited with efficiencies ranging from 78.3 to 100%. The CRISPR-Cpf1 system and the non-homologous end joining mechanism were also investigated, but failed to yield edited strains. We present the first genome editing method for R. eutropha using an electroporation-based CRISPR-Cas9 approach, which significantly increased the efficiency and decreased time to manipulate this facultative chemolithoautotrophic microbe. The novel technique will facilitate more advanced researches and applications of R. eutropha for PHA production and CO 2 conversion.

Electroporation enhances antimicrobial photodynamic therapy mediated by the hydrophobic photosensitizer, hypericin, Electroporation enhances antimicrobial photodynamic inactivation

PubMed Central

de Melo, Wanessa de Cássia Martins Antunes; Lee, Alexander N; Perussi, Janice Rodrigues; Hamblin, Michael R.

2013-01-01

The effective transport of photosensitizers (PS) across the membrane and the intracellular accumulation of PS are the most crucial elements in antimicrobial photodynamic therapy (aPDT). However, due to the morphological complexity of Gram-negative bacteria the penetration of PS is limited, especially hydrophobic PS. Electroporation (EP) could increase the effectiveness of aPDT, by promoting the formation of transient pores that enhance the permeability of the bacterial membrane to PS. In this study we evaluated the combination of aPDT mediated by the hydrophobic PS, hypericin and EP (aPDT/EP) against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. These bacteria were exposed to light (590 nm) in the presence of hypericin (4µM), following electroporation. The results showed that aPDT/EP inactivated 3.67 logs more E. coli and 2.65 logs more S. aureus than aPDT alone. Based on these results we suggest that EP can potentiate the aPDT effect. PMID:24284122

Tutorial: Electroporation of cells in complex materials and tissue

NASA Astrophysics Data System (ADS)

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.

An Efficient Electroporation Protocol for the Genetic Modification of Mammalian Cells

PubMed Central

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

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

PubMed

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.

Electroporation in food processing and biorefinery.

PubMed

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.

Abiotic gene transfer: rare or rampant?

PubMed Central

Kotnik, Tadej; Weaver, James C.

2016-01-01

Phylogenetic studies reveal that horizontal gene transfer (HGT) plays a prominent role in evolution and genetic variability of life. Five biotic mechanisms of HGT among prokaryotic organisms have been extensively characterized: conjugation, competence, transduction, gene-transfer-agent (GTA) particles, and transitory fusion with recombination, but it is not known whether they can account for all natural HGT. It is even less clear how HGT could have occurred before any of these mechanisms had developed. Here, we consider contemporary conditions and experiments on microorganisms to estimate possible roles of abiotic HGT – currently and throughout evolution. Candidate mechanisms include freeze-and-thaw, microbeads-agitation, and electroporation-based transformation, and we posit that these laboratory techniques have analogues in nature acting as mechanisms of abiotic HGT: freeze-and-thaw cycles in polar waters, sand-agitation at foreshores and riverbeds, and lightning-triggered electroporation in near-surface aqueous habitats. We derive conservative order-of-magnitude estimates for rates of microorganisms subjected to freeze-and-thaw cycles, sand-agitation, and lightning-triggered electroporation, at 1024, 1019, and 1017 per year, respectively. Considering the yield of viable transformants, which is by far the highest in electroporation, we argue this may still favor lightning-triggered transformation over the other two mechanisms. Electroporation-based gene transfer also appears to be the most general of these abiotic candidates, and perhaps even of all known HGT mechanisms. Future studies should provide improved estimates of gene transfer rates and cell viability, currently and in the past, but to assess the importance of abiotic HGT in nature, will likely require substantial progress – also in knowledge of biotic HGT. PMID:27067073

An e-learning application on electrochemotherapy

PubMed Central

Corovic, Selma; Bester, Janez; Miklavcic, Damijan

2009-01-01

Background Electrochemotherapy is an effective approach in local tumour treatment employing locally applied high-voltage electric pulses in combination with chemotherapeutic drugs. In planning and performing electrochemotherapy a multidisciplinary expertise is required and collaboration, knowledge and experience exchange among the experts from different scientific fields such as medicine, biology and biomedical engineering is needed. The objective of this study was to develop an e-learning application in order to provide the educational content on electrochemotherapy and its underlying principles and to support collaboration, knowledge and experience exchange among the experts involved in the research and clinics. Methods The educational content on electrochemotherapy and cell and tissue electroporation was based on previously published studies from molecular dynamics, lipid bilayers, single cell level and simplified tissue models to complex biological tissues and research and clinical results of electrochemotherapy treatment. We used computer graphics such as model-based visualization (i.e. 3D numerical modelling using finite element method) and 3D computer animations and graphical illustrations to facilitate the representation of complex biological and physical aspects in electrochemotherapy. The e-learning application is integrated into an interactive e-learning environment developed at our institution, enabling collaboration and knowledge exchange among the users. We evaluated the designed e-learning application at the International Scientific workshop and postgraduate course (Electroporation Based Technologies and Treatments). The evaluation was carried out by testing the pedagogical efficiency of the presented educational content and by performing the usability study of the application. Results The e-learning content presents three different levels of knowledge on cell and tissue electroporation. In the first part of the e-learning application we explain basic principles of electroporation process. The second part provides educational content about importance of modelling and visualization of local electric field in electroporation-based treatments. In the third part we developed an interactive module for visualization of local electric field distribution in 3D tissue models of cutaneous tumors for different parameters such as voltage applied, distance between electrodes, electrode dimension and shape, tissue geometry and electric conductivity. The pedagogical efficiency assessment showed that the participants improved their level of knowledge. The results of usability evaluation revealed that participants found the application simple to learn, use and navigate. The participants also found the information provided by the application easy to understand. Conclusion The e-learning application we present in this article provides educational material on electrochemotherapy and its underlying principles such as cell and tissue electroporation. The e-learning application is developed to provide an interactive educational content in order to simulate the "hands-on" learning approach about the parameters being important for successful therapy. The e-learning application together with the interactive e-learning environment is available to the users to provide collaborative and flexible learning in order to facilitate knowledge exchange among the experts from different scientific fields that are involved in electrochemotherapy. The modular structure of the application allows for upgrade with new educational content collected from the clinics and research, and can be easily adapted to serve as a collaborative e-learning tool also in other electroporation-based treatments such as gene electrotransfer, gene vaccination, irreversible tissue ablation and transdermal gene and drug delivery. The presented e-learning application provides an easy and rapid approach for information, knowledge and experience exchange among the experts from different scientific fields, which can facilitate development and optimisation of electroporation-based treatments. PMID:19843322

An e-learning application on electrochemotherapy.

PubMed

Corovic, Selma; Bester, Janez; Miklavcic, Damijan

2009-10-20

Electrochemotherapy is an effective approach in local tumour treatment employing locally applied high-voltage electric pulses in combination with chemotherapeutic drugs. In planning and performing electrochemotherapy a multidisciplinary expertise is required and collaboration, knowledge and experience exchange among the experts from different scientific fields such as medicine, biology and biomedical engineering is needed. The objective of this study was to develop an e-learning application in order to provide the educational content on electrochemotherapy and its underlying principles and to support collaboration, knowledge and experience exchange among the experts involved in the research and clinics. The educational content on electrochemotherapy and cell and tissue electroporation was based on previously published studies from molecular dynamics, lipid bilayers, single cell level and simplified tissue models to complex biological tissues and research and clinical results of electrochemotherapy treatment. We used computer graphics such as model-based visualization (i.e. 3D numerical modelling using finite element method) and 3D computer animations and graphical illustrations to facilitate the representation of complex biological and physical aspects in electrochemotherapy. The e-learning application is integrated into an interactive e-learning environment developed at our institution, enabling collaboration and knowledge exchange among the users. We evaluated the designed e-learning application at the International Scientific workshop and postgraduate course (Electroporation Based Technologies and Treatments). The evaluation was carried out by testing the pedagogical efficiency of the presented educational content and by performing the usability study of the application. The e-learning content presents three different levels of knowledge on cell and tissue electroporation. In the first part of the e-learning application we explain basic principles of electroporation process. The second part provides educational content about importance of modelling and visualization of local electric field in electroporation-based treatments. In the third part we developed an interactive module for visualization of local electric field distribution in 3D tissue models of cutaneous tumors for different parameters such as voltage applied, distance between electrodes, electrode dimension and shape, tissue geometry and electric conductivity. The pedagogical efficiency assessment showed that the participants improved their level of knowledge. The results of usability evaluation revealed that participants found the application simple to learn, use and navigate. The participants also found the information provided by the application easy to understand. The e-learning application we present in this article provides educational material on electrochemotherapy and its underlying principles such as cell and tissue electroporation. The e-learning application is developed to provide an interactive educational content in order to simulate the "hands-on" learning approach about the parameters being important for successful therapy. The e-learning application together with the interactive e-learning environment is available to the users to provide collaborative and flexible learning in order to facilitate knowledge exchange among the experts from different scientific fields that are involved in electrochemotherapy. The modular structure of the application allows for upgrade with new educational content collected from the clinics and research, and can be easily adapted to serve as a collaborative e-learning tool also in other electroporation-based treatments such as gene electrotransfer, gene vaccination, irreversible tissue ablation and transdermal gene and drug delivery. The presented e-learning application provides an easy and rapid approach for information, knowledge and experience exchange among the experts from different scientific fields, which can facilitate development and optimisation of electroporation-based treatments.

Improvement of electroporation to deliver plasmid DNA into dental follicle cells

PubMed Central

Yao, Shaomian; Rana, Samir; Liu, Dawen; Wise, Gary E.

2010-01-01

Electroporation DNA transfer is a simple and versatile approach to deliver genes. To develop an optimal electroporation protocol to deliver DNA into cells, we conducted square wave electroporation experiments with using rat dental follicle cells as follows: 1) the cells were electroporated at different electric field strengths with lac Z plasmid; 2) plasmid concentrations were tested to determine the optimal doses; 3) various concentrations of bovine serum albumin or fetal bovine serum were added to the pulsing buffer; and, 4) the pulsing durations were studied to determine the optimal duration. These experiments indicated that the optimal electroporation electric field strength was 375 V/cm, and that plasmid concentrations greater than 0.18 μg/μl were required to achieve high transfection efficiency. BSA or FBS in the pulsing buffer significantly improved cell survival and increased the number of transfected cells. The optimal pulsing duration was in the range of 45 to 120 milliseconds (ms) at 375 V/cm. Thus, an improved electroporation protocol was established by optimizing the above parameters. In turn, this electroporation protocol can be used to deliver DNA into dental follicle cells to study the roles of candidate genes in regulating tooth eruption. PMID:19830717

Electroporation in veterinary oncology.

PubMed

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.

Short History of Electroporation for the Study of Developmental Biology

NASA Astrophysics Data System (ADS)

Nakamura, Harukazu

Electroporation at high voltage has been been applied for transformation of bacteria. Since it damages the tissue of higher organisms, it has been limited to bac teria trans formation. Nevertheless, electroporation in higher organisms has been carried out for the transfer of the drugs for treatment of cancer; bleomycin was successfully applied to hepatocellular carcinoma in the Donryu rat (Okino and Mohri, 1987). Mr. Hayakawa (Nepa Gene; Ichikawa, Japan) supplied the electroporator and helped to optimize the condition of electroporation with the BTX electropo rator, and Mr. Imada (Unique Medical-Imada, Natori, Japan) designed and prepared electrodes.

Microfabrication of individual 200 microm diameter transdermal microconduits using high voltage pulsing in salicylic acid and benzoic acid.

PubMed

Ilic, L; Gowrishankar, T R; Vaughan, T E; Herndon, T O; Weaver, J C

2001-01-01

We describe an extension of semiconductor fabrication methods that creates individual approximately 200 microm diameter aqueous pathways through human stratum corneum at predetermined sites. Our hypothesis is that spatially localized electroporation of the multilamellar lipid bilayer membranes provides rapid delivery of salicylic acid to the keratin within corneocytes, leading to localized keratin disruption and then to a microconduit. A microconduit penetrating the isolated stratum corneum supports a volumetric flow of order 0.01 ml per s with a pressure difference of only 0.01 atm (about 10(2) Pa). This study provides a method for rapidly microengineering a pathway in the skin to interface future devices for transdermal drug delivery and sampling of biologically relevant fluids.

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

PubMed

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-learning practical work presented to the students with different professional backgrounds enhanced their knowledge acquired via lectures during EBTT. We compared the learning effect assessed in two experimental groups undergoing the e-learning practical work: electrical engineers and natural scientists. The same level of knowledge on the post-course examination was reached in both groups. The results indicate that our e-learning platform supported by blended learning approach provides an effective learning tool for populations with mixed professional backgrounds and thus plays an important role in bridging the gap between scientific domains involved in electroporation-based technologies and treatments.

Controlled release of optimized electroporation enhances the transdermal efficiency of sinomenine hydrochloride for treating arthritis in vitro and in clinic

PubMed Central

Feng, Shun; Zhu, Lijun; Huang, Zhisheng; Wang, Haojia; Li, Hong; Zhou, Hua; Lu, Linlin; Wang, Ying; Liu, Zhongqiu; Liu, Liang

2017-01-01

Sinomenine hydrochloride (SH) is an ideal drug for the treatment of rheumatoid arthritis and osteoarthritis. However, high plasma concentration of systemically administered SH can release histamine, which can cause rash and gastrointestinal side effects. Topical delivery can increase SH concentration in the synovial fluid without high plasma level, thus minimizing systemic side effects. However, passive diffusion of SH was found to be inefficient because of the presence of the stratum corneum layer. Therefore, an effective method is required to compensate for the low efficiency of SH passive diffusion. In this study, transdermal experiments in vitro and clinical tests were utilized to explore the optimized parameters for electroporation of topical delivery for SH. Fluorescence experiment and hematoxylin and eosin staining analysis were performed to reveal the mechanism by which electroporation promoted permeation. In vitro, optimized electroporation parameters were 3 KHz, exponential waveform, and intensity 10. Using these parameters, transdermal permeation of SH was increased by 1.9–10.1 fold in mice skin and by 1.6–47.1 fold in miniature pig skin compared with passive diffusion. After the electroporation stimulation, the intercellular intervals and epidermal cracks in the skin increased. In clinical tests, SH concentration in synovial fluid was 20.84 ng/mL after treatment with electroporation. Therefore, electroporation with optimized parameters could significantly enhance transdermal permeation of SH. The mechanism by which electroporation promoted permeation was that the electronic pulses made the skin structure looser. To summarize, electroporation may be an effective complementary method for transdermal permeation of SH. The controlled release of electroporation may be a promising clinical method for transdermal drug administration. PMID:28670109

Real-time, in situ monitoring of nanoporation using electric field-induced acoustic signal

NASA Astrophysics Data System (ADS)

Zarafshani, Ali; Faiz, Rowzat; Samant, Pratik; Zheng, Bin; Xiang, Liangzhong

2018-02-01

The use of nanoporation in reversible or irreversible electroporation, e.g. cancer ablation, is rapidly growing. This technique uses an ultra-short and intense electric pulse to increase the membrane permeability, allowing non-permeant drugs and genes access to the cytosol via nanopores in the plasma membrane. It is vital to create a real-time in situ monitoring technique to characterize this process and answer the need created by the successful electroporation procedure of cancer treatment. All suggested monitoring techniques for electroporation currently are for pre-and post-stimulation exposure with no real-time monitoring during electric field exposure. This study was aimed at developing an innovative technology for real-time in situ monitoring of electroporation based on the typical cell exposure-induced acoustic emissions. The acoustic signals are the result of the electric field, which itself can be used in realtime to characterize the process of electroporation. We varied electric field distribution by varying the electric pulse from 1μ - 100ns and varying the voltage intensity from 0 - 1.2ܸ݇ to energize two electrodes in a bi-polar set-up. An ultrasound transducer was used for collecting acoustic signals around the subject under test. We determined the relative location of the acoustic signals by varying the position of the electrodes relative to the transducer and varying the electric field distribution between the electrodes to capture a variety of acoustic signals. Therefore, the electric field that is utilized in the nanoporation technique also produces a series of corresponding acoustic signals. This offers a novel imaging technique for the real-time in situ monitoring of electroporation that may directly improve treatment efficiency.

Current density imaging sequence for monitoring current distribution during delivery of electric pulses in irreversible electroporation.

PubMed

Serša, Igor; Kranjc, Matej; Miklavčič, Damijan

2015-01-01

Electroporation is gaining its importance in everyday clinical practice of cancer treatment. For its success it is extremely important that coverage of the target tissue, i.e. treated tumor, with electric field is within the specified range. Therefore, an efficient tool for the electric field monitoring in the tumor during delivery of electroporation pulses is needed. The electric field can be reconstructed by the magnetic resonance electric impedance tomography method from current density distribution data. In this study, the use of current density imaging with MRI for monitoring current density distribution during delivery of irreversible electroporation pulses was demonstrated. Using a modified single-shot RARE sequence, where four 3000 V and 100 μs long pulses were included at the start, current distribution between a pair of electrodes inserted in a liver tissue sample was imaged. Two repetitions of the sequence with phases of refocusing radiofrequency pulses 90° apart were needed to acquire one current density image. For each sample in total 45 current density images were acquired to follow a standard protocol for irreversible electroporation where 90 electric pulses are delivered at 1 Hz. Acquired current density images showed that the current density in the middle of the sample increased from first to last electric pulses by 60%, i.e. from 8 kA/m2 to 13 kA/m2 and that direction of the current path did not change with repeated electric pulses significantly. The presented single-shot RARE-based current density imaging sequence was used successfully to image current distribution during delivery of short high-voltage electric pulses. The method has a potential to enable monitoring of tumor coverage by electric field during irreversible electroporation tissue ablation.

Gene Suppression of Mouse Testis In Vivo Using Small Interfering RNA Derived from Plasmid Vectors

PubMed Central

Takizawa, Takami; Ishikawa, Tomoko; Kosuge, Takuji; Mizuguchi, Yoshiaki; Sato, Yoko; Koji, Takehiko; Araki, Yoshihiko; Takizawa, Toshihiro

2012-01-01

We evaluated whether inhibiting gene expression by small interfering RNA (siRNA) can be used for an in vivo model using a germ cell-specific gene (Tex101) as a model target in mouse testis. We generated plasmid-based expression vectors of siRNA targeting the Tex101 gene and transfected them into postnatal day 10 mouse testes by in vivo electroporation. After optimizing the electroporation conditions using a vector transfected into the mouse testis, a combination of high- and low-voltage pulses showed excellent transfection efficiency for the vectors with minimal tissue damage, but gene suppression was transient. Gene suppression by in vivo electroporation may be helpful as an alternative approach when designing experiments to unravel the basic role of testicular molecules. PMID:22489107

Use of electroporation to study the cytotoxic effects of fluorodeoxyuridylate in intact cells.

PubMed

Jastreboff, M M; Sokoloski, J A; Bertino, J R; Narayanan, R

1987-04-15

The introduction of 2'-deoxyuridine 5'-monophosphate and its analog, 5-fluoro-2'-deoxyuridine 5'-monophosphate, into intact CCRF-CEM and NIH3T3 cells was achieved by electroporation. Following electroporation, cells were shown to be fully functional as monitored by the incorporation of deoxyuridylate, after conversion to thymidylate, into DNA. Pretreatment of cells with fluorodeoxyuridine completely abolished this effect. In contrast, introduction of the fluoro analog into cells by electroporation markedly inhibited both DNA synthesis and cell growth in a time-dependent manner. Thus, electroporation offers a powerful tool to permeabilize cells to a variety of cellular metabolites and antimetabolites.

Membrane permeabilization of mammalian cells using bursts of high magnetic field pulses

PubMed Central

Grainys, Audrius; Kranjc, Matej; Miklavčič, Damijan

2017-01-01

Background Cell membrane permeabilization by pulsed electromagnetic fields (PEMF) is a novel contactless method which results in effects similar to conventional electroporation. The non-invasiveness of the methodology, independence from the biological object homogeneity and electrical conductance introduce high flexibility and potential applicability of the PEMF in biomedicine, food processing, and biotechnology. The inferior effectiveness of the PEMF permeabilization compared to standard electroporation and the lack of clear description of the induced transmembrane transport are currently of major concern. Methods The PEMF permeabilization experiments have been performed using a 5.5 T, 1.2 J pulse generator with a multilayer inductor as an applicator. We investigated the feasibility to increase membrane permeability of Chinese Hamster Ovary (CHO) cells using short microsecond (15 µs) pulse bursts (100 or 200 pulses) at low frequency (1 Hz) and high dB/dt (>106 T/s). The effectiveness of the treatment was evaluated by fluorescence microscopy and flow cytometry using two different fluorescent dyes: propidium iodide (PI) and YO-PRO®-1 (YP). The results were compared to conventional electroporation (single pulse, 1.2 kV/cm, 100 µs), i.e., positive control. Results The proposed PEMF protocols (both for 100 and 200 pulses) resulted in increased number of permeable cells (70 ± 11% for PI and 67 ± 9% for YP). Both cell permeabilization assays also showed a significant (8 ± 2% for PI and 35 ± 14% for YP) increase in fluorescence intensity indicating membrane permeabilization. The survival was not affected. Discussion The obtained results demonstrate the potential of PEMF as a contactless treatment for achieving reversible permeabilization of biological cells. Similar to electroporation, the PEMF permeabilization efficacy is influenced by pulse parameters in a dose-dependent manner. PMID:28462057

GMP-Grade mRNA Electroporation of Dendritic Cells for Clinical Use.

PubMed

Derdelinckx, Judith; Berneman, Zwi N; Cools, Nathalie

2016-01-01

mRNA-electroporated dendritic cells (DC) are demonstrating clinical benefit in patients in many therapeutic areas, including cancer and infectious diseases. According to current good manufacturing guidelines, cell-based medicinal products have to be defined for identity, purity, potency, stability, and viability. In order to comply with the directives and guidelines defined by the regulatory authorities, we report here a standardized and reproducible method for the manufacturing of clinical-grade mRNA-transfected DC.

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

PubMed

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.

Combining Electrolysis and Electroporation for Tissue Ablation.

PubMed

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. © The Author(s) 2014.

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

PubMed

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.

Transformation of Rhodococcus fascians by High-Voltage Electroporation and Development of R. fascians Cloning Vectors

PubMed Central

Desomer, Jan; Dhaese, Patrick; Montagu, Marc Van

1990-01-01

The analysis of the virulence determinants of phytopathogenic Rhodococcus fascians has been hampered by the lack of a system for introducing exogenous DNA. We investigated the possibility of genetic transformation of R. fascians by high-voltage electroporation of intact bacterial cells in the presence of plasmid DNA. Electrotransformation in R. fascians D188 resulted in transformation frequencies ranging from 105/μg of DNA to 107/μg of DNA, depending on the DNA concentration. The effects of different electrical parameters and composition of electroporation medium on transformation efficiency are presented. By this transformation method, a cloning vector (pRF28) for R. fascians based on an indigenous 160-kilobase (chloramphenicol and cadmium resistance-encoding) plasmid pRF2 from strain NCPPB 1675 was developed. The origin of replication and the chloramphenicol resistance gene on pRF28 were used to construct cloning vectors that are capable of replication in R. fascians and Escherichia coli. The electroporation method presented was efficient enough to allow detection of the rare integration of replication-deficient pRF28 derivatives in the R. fascians D188 genome via either homologous or illegitimate recombination. Images PMID:16348290

Developing a de novo targeted knock-in method based on in utero electroporation into the mammalian brain.

PubMed

Tsunekawa, Yuji; Terhune, Raymond Kunikane; Fujita, Ikumi; Shitamukai, Atsunori; Suetsugu, Taeko; Matsuzaki, Fumio

2016-09-01

Genome-editing technology has revolutionized the field of biology. Here, we report a novel de novo gene-targeting method mediated by in utero electroporation into the developing mammalian brain. Electroporation of donor DNA with the CRISPR/Cas9 system vectors successfully leads to knock-in of the donor sequence, such as EGFP, to the target site via the homology-directed repair mechanism. We developed a targeting vector system optimized to prevent anomalous leaky expression of the donor gene from the plasmid, which otherwise often occurs depending on the donor sequence. The knock-in efficiency of the electroporated progenitors reached up to 40% in the early stage and 20% in the late stage of the developing mouse brain. Furthermore, we inserted different fluorescent markers into the target gene in each homologous chromosome, successfully distinguishing homozygous knock-in cells by color. We also applied this de novo gene targeting to the ferret model for the study of complex mammalian brains. Our results demonstrate that this technique is widely applicable for monitoring gene expression, visualizing protein localization, lineage analysis and gene knockout, all at the single-cell level, in developmental tissues. © 2016. Published by The Company of Biologists Ltd.

Direct and efficient transfection of mouse neural stem cells and mature neurons by in vivo mRNA electroporation.

PubMed

Bugeon, Stéphane; de Chevigny, Antoine; Boutin, Camille; Coré, Nathalie; Wild, Stefan; Bosio, Andreas; Cremer, Harold; Beclin, Christophe

2017-11-01

In vivo brain electroporation of DNA expression vectors is a widely used method for lineage and gene function studies in the developing and postnatal brain. However, transfection efficiency of DNA is limited and adult brain tissue is refractory to electroporation. Here, we present a systematic study of mRNA as a vector for acute genetic manipulation in the developing and adult brain. We demonstrate that mRNA electroporation is far more efficient than DNA electroporation, and leads to faster and more homogeneous protein expression in vivo Importantly, mRNA electroporation allows the manipulation of neural stem cells and postmitotic neurons in the adult brain using minimally invasive procedures. Finally, we show that this approach can be efficiently used for functional studies, as exemplified by transient overexpression of the neurogenic factor Myt1l and by stably inactivating Dicer nuclease in vivo in adult born olfactory bulb interneurons and in fully integrated cortical projection neurons. © 2017. Published by The Company of Biologists Ltd.

Modelling in conventional electroporation for model cell with organelles using COMSOL Multiphysics

NASA Astrophysics Data System (ADS)

Sulaeman, M. Y.; Widita, R.

2016-03-01

Conventional electroporation is a formation of pores in the membrane cell due to the external electric field applied to the cell. The purpose of creating pores in the cell using conventional electroporation are to increase the effectiveness of chemotherapy (electrochemotherapy) and to kill cancer tissue using irreversible electroporation. Modeling of electroporation phenomenon on a model cell had been done by using software COMSOL Multiphysics 4.3b with the applied external electric field with intensity at 1.1 kV/cm to find transmembrane voltage and pore density. It can be concluded from the results of potential distribution and transmembrane voltage, it show that pores formation only occurs in the membrane cells and it could not penetrate into inside the model cell so there is not pores formation in its organells.

Pulsed Electromagnetic Field Assisted in vitro Electroporation: A Pilot Study

NASA Astrophysics Data System (ADS)

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.

Pulsed Electromagnetic Field Assisted in vitro Electroporation: A Pilot Study

PubMed Central

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

2016-01-01

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. PMID:27634482

Identification and Application of Plasmids Suitable for Transfer of Foreign DNA to Members of the Genus Gordonia

PubMed Central

Arenskötter, Matthias; Baumeister, Dirk; Kalscheuer, Rainer; Steinbüchel, Alexander

2003-01-01

Gene transfer systems for Gordonia polyisoprenivorans strains VH2 and Y2K based on electroporation and conjugation, respectively, were established. Several parameters were optimized, resulting in transformation efficiencies of >4 × 105 CFU/μg of plasmid DNA. In contrast to most previously described electroporation protocols, the highest efficiencies were obtained by applying a heat shock after the intrinsic electroporation. Under these conditions, transfer and autonomous replication of plasmid pNC9503 was also demonstrated to proceed in G. alkanivorans DSM44187, G. nitida DSM44499T, G. rubropertincta DSM43197T, G. rubropertincta DSM46038, and G. terrae DSM43249T. Conjugational plasmid DNA transfer to G. polyisoprenivorans resulted in transfer frequencies of up to 5 × 10−6 of the recipient cells. Recombinant strains capable of polyhydroxyalkanoate synthesis from alkanes were constructed. PMID:12902293

Evaluating the potential of poly(beta-amino ester) nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells.

PubMed

Bhise, Nupura S; Wahlin, Karl J; Zack, Donald J; Green, Jordan J

2013-01-01

Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts. A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling. 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacry-late-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available reagents, including Lipofectamine® 2000, FuGENE® HD, and 25 kDa branched polyethylenimine, for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa, and enabled coexpression of exogenously delivered genes, as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation, but not by poly(beta-amino ester) reprogramming, could be differentiated toward the neuronal lineage, specifically pseudostratified optic cups. This study shows that certain nonviral reprogramming methods may not necessarily be safer than viral approaches and that maximizing exogenous gene expression of reprogramming factors is not sufficient to ensure successful reprogramming.

Ablative therapies: Advantages and disadvantages of radiofrequency, cryotherapy, microwave and electroporation methods, or how to choose the right method for an individual patient?

PubMed

Seror, O

2015-06-01

Several ablation techniques are currently available. Except for electroporation, all of these methods cause fatal damage at a cellular level and irreversible architectural deconstruction at a tissue level by thermal effects. Ablation of a tumor using one of these techniques, whether thermal or otherwise, requires applicators to be positioned from which the energy is delivered in situ. Some techniques, however, require several applicators to be inserted (multibipolar radiofrequency, cryotherapy and electroporation) whereas a single applicator is often sufficient with other technologies (monopolar radiofrequency and microwave). These methods are conceptually very similar but are distinguished from each other in practice through the technologies they use. It is essential to understand these differences as they influence the advantages and limitations of each of the techniques. There is no such thing as the perfect multifunctional ablation device and choice is dictated on an individual patient basis depending on the aim of treatment, which itself depends on each patient's clinical situation. Copyright © 2015 Éditions françaises de radiologie. Published by Elsevier Masson SAS. All rights reserved.

Transdermal transport pathway creation: Electroporation pulse order.

PubMed

Becker, Sid; Zorec, Barbara; Miklavčič, Damijan; Pavšelj, Nataša

2014-11-01

In this study we consider the physics underlying electroporation which is administered to skin in order to radically increase transdermal drug delivery. The method involves the application of intense electric fields to alter the structure of the impermeable outer layer, the stratum corneum. A generally held view in the field of skin electroporation is that the skin's drop in resistance (to transport) is proportional to the total power of the pulses (which may be inferred by the number of pulses administered). Contrary to this belief, experiments conducted in this study show that the application of high voltage pulses prior to the application of low voltage pulses result in lower transport than when low voltage pulses alone are applied (when less total pulse power is administered). In order to reconcile these unexpected experimental results, a computational model is used to conduct an analysis which shows that the high density distribution of very small aqueous pathways through the stratum corneum associated with high voltage pulses is detrimental to the evolution of larger pathways that are associated with low voltage pulses. Copyright © 2014 Elsevier Inc. All rights reserved.

The effects of metallic implants on electroporation therapies: feasibility of irreversible electroporation for brachytherapy salvage.

PubMed

Neal, Robert E; Smith, Ryan L; Kavnoudias, Helen; Rosenfeldt, Franklin; Ou, Ruchong; Mclean, Catriona A; Davalos, Rafael V; Thomson, Kenneth R

2013-12-01

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. 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. 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. 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.

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

NASA Astrophysics Data System (ADS)

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 techniques. Cells were patterned on the nanochannel array and collectively were electroporated in parallel, injected with cargo in Z-direction. Controlling the dose was demonstrated with the external pulse durations at high-throughput. The 'electrophoretic'- expedited delivery of large molecular weight plasmids were demonstrated with large numbers of primary cells simultaneously, which cannot be achieved in BEP and MEP. Two clinically valuable case studies were performed with our 3D NEP for living cell sensing / interrogation. (1) In the case of in vitro transfection of primary cardiomyocytes, we studied the dose-effects of miR-29 on mitochondrial changes and the suppression of the Mcl-1 gene in adult mouse cardiomyocytes by precisely controlling the miR-29 dose injected. (2) Glioma stem cells (GSCs), a type of cell hypothesized to be highly aggressive and to lead to the relapses of gliobastoma in human brain, was studied at single cell resolution on 3D NEP platform. The developed 3D NEP system moves towards clinically oriented and user-friendly tools for life science applications. The batch-treated cells with controlled dosage delivery provide a useful tool for single cell analysis. The pioneering experiments in this work have demonstrated the 3D NEP for the applications of cell reprogramming, adoptive immunotherapy, in vitro cardiomyocytes transfection and glioma stem cells study.

Cationic solid-lipid nanoparticles are as efficient as electroporation in DNA vaccination against visceral leishmaniasis in mice.

PubMed

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. © 2013 John Wiley & Sons Ltd.

Microfluidic Screening of Electric Fields for Electroporation

PubMed Central

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

2016-01-01

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. PMID:26893024

Single cell electroporation using proton beam fabricated biochips

NASA Astrophysics Data System (ADS)

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.

Comparative evaluation of transmembrane ion transport due to monopolar and bipolar nanosecond, high-intensity electroporation pulses based on full three-dimensional analyses

NASA Astrophysics Data System (ADS)

Hu, Q.; Joshi, R. P.

2017-07-01

Electric pulse driven membrane poration finds applications in the fields of biomedical engineering and drug/gene delivery. Here we focus on nanosecond, high-intensity electroporation and probe the role of pulse shape (e.g., monopolar-vs-bipolar), multiple electrode scenarios, and serial-versus-simultaneous pulsing, based on a three-dimensional time-dependent continuum model in a systematic fashion. Our results indicate that monopolar pulsing always leads to higher and stronger cellular uptake. This prediction is in agreement with experimental reports and observations. It is also demonstrated that multi-pronged electrode configurations influence and increase the degree of cellular uptake.

[Markerless DNA deletion based on Red recombination and in vivo I-Sec I endonuclease cleavage in Escherichia coli chromosome].

PubMed

Zhu, Meiqin; Yu, Jian; Zhou, Changlin; Fang, Hongqing

2016-01-01

Red-based recombineering has been widely used in Escherichia coli genome modification through electroporating PCR fragments into electrocompetent cells to replace target sequences. Some mutations in the PCR fragments may be brought into the homologous regions near the target. To solve this problem in markeless gene deletion we developed a novel method characterized with two-step recombination and a donor plasmid. First, generated by PCR a linear DNA cassette which comprises a I-Sec I site-containing marker gene and homologous arms was electroporated into cells for marker-substitution deletion of the target sequence. Second, after a donor plasmid carrying the I-Sec I site-containing fusion homologous arm was chemically transformed into the marker-containing cells, the fusion arms and the marker was simultaneously cleaved by I-Sec I endonuclease and the marker-free deletion was stimulated by double-strand break-mediated intermolecular recombination. Eleven nonessential regions in E. coli DH1 genome were sequentially deleted by our method, resulting in a 10.59% reduced genome size. These precise deletions were also verified by PCR sequencing and genome resequencing. Though no change in the growth rate on the minimal medium, we found the genome-reduced strains have some alteration in the acid resistance and for the synthesis of lycopene.

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

PubMed Central

Brown, Patricia A; Bodles-Brakhop, Angela M; Pope, Melissa A; Draghia-Akli, Ruxandra

2009-01-01

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. PMID:19149896

A pliable electroporation patch (ep-Patch) for efficient delivery of nucleic acid molecules into animal tissues with irregular surface shapes.

PubMed

Wei, Zewen; Huang, Yuanyu; Zhao, Deyao; Hu, Zhiyuan; Li, Zhihong; Liang, Zicai

2015-01-05

Delivery of nucleic acids into animal tissues by electroporation is an appealing approach for various types of gene therapy, but efficiency of existing methodsis not satisfactory. Here we present the validation of novel electroporation patch (ep-Patch) for efficient delivery of DNA and siRNA into mouse tissues. Using micromachining technology, closely spaced gold electrodes were made on the pliable parylene substrate to form a patch-like electroporation metrics. It enabled large coverage of the target tissues and close surface contact between the tissues and electrodes, thus providing a uniform electric field to deliver nucleic acids into tissues, even beneath intact skin. Using this ep-Patch for efficiently delivery of both DNA and siRNA, non-invasive electroporation of healthy mouse muscle tissue was successfully achieved. Delivery of these nucleic acids was performed to intact tumors with satisfactory results. Silencing of tumor genes using the ep-Patch was also demonstrated on mice. This pliable electroporation patch method constitutes a novel way of in vivo delivery of siRNA and DNA to certain tissues or organs to circumvent the disadvantages of existing methodologies for in vivo delivery of nucleic acid molecules.

Perspectives on Transdermal Electroporation

PubMed Central

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

In vitro analysis of various cell lines responses to electroporative electric pulses by means of electrical impedance spectroscopy.

PubMed

García-Sánchez, Tomás; Bragós, Ramon; Mir, Lluis M

2018-06-07

This paper reports the comparative analysis, by means of electric impedance spectroscopy measurements, of three different cell lines subjected to electroporative pulses. The multifrequency information is recorded simultaneously at 21 frequency values in the range between 5 kHz and 1.3 MHz using a multisine based measuring approach. The analysis of the pre-electroporation impedance spectra shows how the system is able to detect differences and similarities between the cell lines under analysis. Particularly, a good agreement is found between the average cell diameter and the characteristic frequency (the frequency corresponding to a maximum in the imaginary part of the impedance). The measurements performed during electroporation at three different electric field intensities show how the impedance spectra changes dynamically between the consecutive pulses of a train of 8,100 µs pulses delivered at 1 Hz repetition rate. There are clear differences between the changes in the impedance measured at low and high frequency. The multifrequency information has been fitted to an electrical equivalent model in order to understand the different contributions in the observed impedance changes (mainly separate between membrane permeabilization and the conductivity changes in the extracellular medium). Finally, a ratio of the low and high frequency impedance information is used to estimate the accumulated impedance decay and to compare it to the internalization of a fluorescent permeabilization reporter. The comparison between both techniques at the three electroporation electric field intensities assayed confirms the ability of impedance measurements to detect in a precise way the level of membrane permeabilization. Additionally, this study demonstrates how the real time information obtained thanks to impedance measurements can provide a more precise quantification of the membrane permeabilization extent. Copyright © 2018 Elsevier B.V. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Neal, Robert E., E-mail: robert.neal@alfred.org.au; Smith, Ryan L., E-mail: ryan.smith@wbrc.org.au; Kavnoudias, Helen, E-mail: H.Kavnoudias@alfred.org.au

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 expiredmore » 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.« less

Poloxamer 188 decreases susceptibility of artificial lipid membranes to electroporation.

PubMed Central

Sharma, V; Stebe, K; Murphy, J C; Tung, L

1996-01-01

The effect of a nontoxic, nonionic block co-polymeric surface active agent, poloxamer 188, on electroporation of artificial lipid membranes made of azolectin, was investigated. Two different experimental protocols were used in our study: charge pulse and voltage clamp. For the charge pulse protocol, membranes were pulsed with a 10-micronsecond rectangular voltage waveform, after which membrane voltage decay was observed through an external 1-M omega resistance. For the voltage clamp protocol the membranes were pulsed with a waveform that consisted of an initial 10-microsecond rectangular phase, followed by a negative sloped ramp that decayed to zero in the subsequent 500 microseconds. Several parameters characterizing the electroporation process were measured and compared for the control membranes and membranes treated with 1.0 mM poloxamer 188. For both the charge pulse and voltage clamp experiments, the threshold voltage (amplitude of initial rectangular phase) and latency time (time elapsed between the end of rectangular phase and the onset of membrane electroporation) were measured. Membrane conductance (measured 200 microseconds after the initial rectangular phase) and rise time (tr; the time required for the porated membrane to reach a certain conductance value) were also determined for the voltage clamp experiments, and postelectroporation time constant (PE tau; the time constant for transmembrane voltage decay after onset of electroporation) for the charge pulse experiments. The charge pulse experiments were performed on 23 membranes with 10 control and 13 poloxamer-treated membranes, and voltage pulse experiments on 49 membranes with 26 control and 23 poloxamer-treated membranes. For both charge pulse and voltage clamp experiments, poloxamer 188-treated membranes exhibited a statistically higher threshold voltage (p = 0.1 and p = 0.06, respectively), and longer latency time (p = 0.04 and p = 0.05, respectively). Also, poloxamer 188-treated membranes were found to have a relatively lower conductance (p = 0.001), longer time required for the porated membrane to reach a certain conductance value (p = 0.05), and longer postelectroporation time constant (p = 0.005). Furthermore, addition of poloxamer 188 was found to reduce the membrane capacitance by approximately 4-8% in 5 min. These findings suggest that poloxamer 188 adsorbs into the lipid bilayers, thereby decreasing their susceptibility to electroporation. Images FIGURE 1 PMID:8968593

Preventing Scars after Injury with Partial Irreversible Electroporation

PubMed Central

Golberg, Alexander; Villiger, Martin; Khan, Saiqa; Quinn, Kyle P.; Lo, William C. Y.; Bouma, Brett E.; Mihm, Martin C.; Austen, William G.; Yarmush, Martin L.

2017-01-01

Preventing the formation of hypertrophic scars, especially those that are a result of major trauma or burns, would have enormous impact in the fields of regenerative and trauma medicine. In this report, we introduce a non-invasive method to prevent scarring based on non-thermal partial irreversible electroporation. Contact burn injuries in rats were treated with varying treatment parameters to optimize the treatment protocol. Scar surface area and structural properties of the scar were assessed with histology and non-invasive, longitudinal imaging with polarization-sensitive optical coherence tomography. We found that partial irreversible electroporation using 200 pulses of 250 V and 70 μs duration, delivered at 3 Hz every 20 days during a total of five therapy sessions after the initial burn injury resulted in a 57.9% reduction of the scar area in comparison with untreated scars and structural features approaching those of normal skin. Noteworthy, unlike humans, rats do not develop hypertrophic scars. Therefore, the use of a rat animal model is the limiting factor of this work. PMID:27393126

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

NASA Astrophysics Data System (ADS)

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

2012-07-01

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.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. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr30578j

Macroscopic Modeling of In Vivo Drug Transport in Electroporated Tissue.

PubMed

Boyd, Bradley; Becker, Sid

2016-03-01

This study develops a macroscopic model of mass transport in electroporated biological tissue in order to predict the cellular drug uptake. The change in the macroscopic mass transport coefficient is related to the increase in electrical conductivity resulting from the applied electric field. Additionally, the model considers the influences of both irreversible electroporation (IRE) and the transient resealing of the cell membrane associated with reversible electroporation. Two case studies are conducted to illustrate the applicability of this model by comparing transport associated with two electrode arrangements: side-by-side arrangement and the clamp arrangement. The results show increased drug transmission to viable cells is possible using the clamp arrangement due to the more uniform electric field.

The Influence of Vesicle Shape and Medium Conductivity on Possible Electrofusion under a Pulsed Electric Field

PubMed Central

Liu, Linying; Mao, Zheng; Zhang, Jianhua; Liu, Na; Liu, Qing Huo

2016-01-01

The effects of electric field on lipid membrane and cells have been extensively studied in the last decades. The phenomena of electroporation and electrofusion are of particular interest due to their wide use in cell biology and biotechnology. However, numerical studies on the electrofusion of cells (or vesicles) with different deformed shapes are still rare. Vesicle, being of cell size, can be treated as a simple model of cell to investigate the behaviors of cell in electric field. Based on the finite element method, we investigate the effect of vesicle shape on electrofusion of contact vesicles in various medium conditions. The transmembrane voltage (TMV) and pore density induced by a pulsed field are examined to analyze the possibility of vesicle fusion. In two different medium conditions, the prolate shape is observed to have selective electroporation at the contact area of vesicles when the exterior conductivity is smaller than the interior one; selective electroporation is more inclined to be found at the poles of the oblate vesicles when the exterior conductivity is larger than the interior one. Furthermore, we find that when the exterior conductivity is lower than the internal conductivity, the pulse can induce a selective electroporation at the contact area between two vesicles regardless of the vesicle shape. Both of these two findings have important practical applications in guiding electrofusion experiments. PMID:27391692

An Efficient System for Gene Perturbation in Embryonic Hippocampal Progenitors Using Ex Vivo Electroporation Followed by In Vitro Dissociated Cell Culture

PubMed Central

Muralidharan, Bhavana

2018-01-01

We established an efficient cell culture assay that permits combinatorial genetic perturbations in hippocampal progenitors to examine cell-autonomous mechanisms of fate specification. The procedure begins with ex vivo electroporation of isolated, intact embryonic brains, in a manner similar to in utero electroporation but with greatly improved access and targeting. The electroporated region is then dissected and transiently maintained in organotypic explant culture, followed by dissociation and plating of cells on coverslips for in vitro culture. This assay recapitulates data obtained in vivo with respect to the neuron-glia cell fate switch and can be effectively used to test intrinsic or extrinsic factors that regulate this process. The advantages of this ex vivo procedure over in utero electroporation include the fact that distinct combinations of perturbative reagents can be introduced in different embryos from a single litter, and issues related to embryonic lethality of transgenic animals can be circumvented. PMID:29760561

A phase 1, randomized, controlled dose-escalation study of EP-1300 polyepitope DNA vaccine against Plasmodium falciparum malaria administered via electroporation.

PubMed

Spearman, Paul; Mulligan, Mark; Anderson, Evan J; Shane, Andi L; Stephens, Kathy; Gibson, Theda; Hartwell, Brooke; Hannaman, Drew; Watson, Nora L; Singh, Karnail

2016-11-04

Plasmodium falciparum malaria is one of the leading infectious causes of childhood mortality in Africa. EP-1300 is a polyepitope plasmid DNA vaccine expressing 38 cytotoxic T cell epitopes and 16 helper T cell epitopes derived from P. falciparum antigens expressed predominantly in the liver phase of the parasite's life cycle. We performed a phase 1 randomized, placebo-controlled, dose escalation clinical trial of the EP-1300 DNA vaccine administered via electroporation using the TriGrid Delivery System device (Ichor Medical Systems). Although the delivery of the EP-1300 DNA vaccine via electroporation was safe, tolerability was less than that usually observed with standard needle and syringe intramuscular administration. This was primarily due to acute local discomfort at the administration site during electroporation. Despite the use of electroporation, the vaccine was poorly immunogenic. The reasons for the poor immunogenicity of this polyepitope DNA vaccine remain uncertain. ClinicalTrials.gov NCT01169077. Copyright © 2016 Elsevier Ltd. All rights reserved.

An Efficient System for Gene Perturbation in Embryonic Hippocampal Progenitors Using Ex Vivo Electroporation Followed by In Vitro Dissociated Cell Culture.

PubMed

Muralidharan, Bhavana; D'Souza, Leora; Tole, Shubha

2018-01-01

We established an efficient cell culture assay that permits combinatorial genetic perturbations in hippocampal progenitors to examine cell-autonomous mechanisms of fate specification. The procedure begins with ex vivo electroporation of isolated, intact embryonic brains, in a manner similar to in utero electroporation but with greatly improved access and targeting. The electroporated region is then dissected and transiently maintained in organotypic explant culture, followed by dissociation and plating of cells on coverslips for in vitro culture. This assay recapitulates data obtained in vivo with respect to the neuron-glia cell fate switch and can be effectively used to test intrinsic or extrinsic factors that regulate this process. The advantages of this ex vivo procedure over in utero electroporation include the fact that distinct combinations of perturbative reagents can be introduced in different embryos from a single litter, and issues related to embryonic lethality of transgenic animals can be circumvented.

The Safety and Efficacy of Irreversible Electroporation for Large Hepatocellular Carcinoma.

PubMed

Zeng, Jianying; Liu, Guifeng; Li, Zhong-Hai; Yang, Yi; Fang, Gang; Li, Rong-Rong; Xu, Ke-Cheng; Niu, Lizhi

2017-02-01

This study aimed to investigate the safety and effectiveness of irreversible electroporation ablation for unresectable large liver cancer. Fourteen patients were enrolled: 8 with large hepatocellular carcinoma (tumor diameter: 5.1-11.5 cm) and 6 with medium hepatocellular carcinoma (tumor diameter: 3.0-4.1 cm). All patients received percutaneous irreversible electroporation ablation. Ablation time and the incidence of complications were assessed by a t test. Post-irreversible electroporation and regular contrast-enhanced computerized tomography scans were performed to investigate the effect of tumor size (large vs medium) on irreversible electroporation treatment efficacy; 4-table data were assessed using a Fisher exact test. The 14 patients completed irreversible electroporation ablation successfully. In the large hepatocellular carcinoma group, no major complications occurred in the perioperative period. Minor complications comprised bloating, hypokalemia, edema, low white blood cells, and blood clotting abnormalities. All complications were mild and improved after symptomatic treatment. The frequency of minor complications was not significantly different ( P > .05) compared with the medium hepatocellular carcinoma group. The average follow-up time was 2.8 ± 2.1 months and complete ablation was achieved in 25% (2/8; residual = 75%). For the patients with medium hepatocellular carcinoma, the mean follow-up time was 4.3 ± 3.2 months; the rate of complete ablation was 66.6% (4/6; residual rate = 33.3%). The complete ablation rate was not statistically different between the 2 groups ( P > .05). Irreversible electroporation ablation for unresectable large hepatocellular carcinoma is safe, with no major complications. Short-term efficacy is relatively good; however, long-term efficacy remains to be explored.

In vivo gene delivery to the postnatal ferret cerebral cortex by DNA electroporation.

PubMed

Borrell, Víctor

2010-02-15

Ferrets have been extensively used to unravel the neural mechanisms of coding and processing of visual information, and also to identify the developmental mechanisms underlying the emergence of such a complex and fine-tuned neural system. In recent years numerous tools have been generated that allow studying neural systems with unprecedented power. Unfortunately, because many of these tools are genetically encoded, they are having a limited impact on research involving "non-genetic" species, like ferret, cat and monkey. Here I show how in vivo electroporation can be performed in postnatal ferret kits to deliver genetic constructs to pyramidal neurons of the cerebral cortex. Electroporation of GFP- and DsRed-encoding plasmids results in labeling of cortical progenitors first, then migrating neurons, and finally differentiating neurons and their processes. This technique also allows for the genetic manipulation of cortical development in the ferret, as illustrated by electroporation of a dominant-negative form of Cdk5. In the mature brain of electroporated animals, expression of reporter genes reveals the detailed morphological traits of cortical pyramids, including their axonal and dendritic arborization, and dendritic spines. I also show that postnatal electroporation can be used for the transfection of a massive cortical territory, or it can be specifically directed to a subset of cortical areas, and even only to a few scattered pyramids along the cortical mantle. In vivo electroporation of postnatal ferrets is therefore an effective, rapid, simple and highly versatile method for delivering genetic constructs to this animal, optimal for both developmental studies and adult anatomical/functional studies. Copyright 2009 Elsevier B.V. All rights reserved.

Educational application for visualization and analysis of electric field strength in multiple electrode electroporation.

PubMed

Mahnič-Kalamiza, Samo; Kotnik, Tadej; Miklavčič, Damijan

2012-10-30

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. 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. In this paper we outline the need and offer a solution in medical education in the field of electroporation-based treatments, e.g. primarily electrochemotherapy and non-thermal irreversible tissue ablation. We present the background, the means of implementation and the fully functional application, which is the first of its kind. While the initial feedback from students that have evaluated this application as part of an e-learning course is positive, a formal study is planned to thoroughly evaluate the current version and identify possible future improvements and modifications.

Simulation of micro/nano electroporation for cell transfection

NASA Astrophysics Data System (ADS)

Zhang, Guocheng; Fan, Na; Jiang, Hai; Guo, Jian; Peng, Bei

2018-03-01

The 3D micro/nano electroporation for transfection has become a powerful biological cell research technique with the development of micro-nano manufacturing technology. The micro channels connected the cells with transfection reagents on the chip were important to the transmemnbrane potentical, which directly influences the electroporation efficiency. In this study, a two-dimensional model for electroporation of cells was designed to address the effects of channels’ sizes and number on transmembrane potential. The simulation results indicated that the transmembrane potential increased with increasing size of channels’ entrances. Moreover, compared with single channel entrance, the transmembrane potential was higher when the cells located at multiple channels entrances. These results suggest that it IS required to develop higher micro manufacturing technology to create channels as we expected size.

Use of electroporation for high-molecular-weight DNA-mediated gene transfer.

PubMed

Jastreboff, M M; Ito, E; Bertino, J R; Narayanan, R

1987-08-01

Electroporation was used to introduce high-molecular-weight DNA into murine hematopoietic cells and NIH3T3 cells. CCRF-CEM cells were stably transfected with SV2NEO plasmid and the genomic DNA from G-418-resistant clones (greater than 65 kb) was introduced into mouse bone marrow and NIH3T3 cells by electroporation. NEO sequences and expression were detected in the hematopoietic tissues of lethally irradiated mice, with 24% of individual spleen colonies expressing NEO. The frequency of genomic DNA transfer into NIH3T3 cells was 0.25 X 10(-3). Electroporation thus offers a powerful mode of gene transfer not only of cloned genes but also of high-molecular-weight DNA into cells.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sommer, C. M., E-mail: christof.sommer@med.uni-heidelberg.de; Fritz, S., E-mail: stefan.fritz@med.uni-heidelberg.de; Vollherbst, D., E-mail: dominikvollherbst@web.de

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 killedmore » 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.« less

Electroporation enhances mitomycin C cytotoxicity on T24 bladder cancer cell line: a potential improvement of intravesical chemotherapy in bladder cancer.

PubMed

Vásquez, Juan L; Gehl, Julie; Hermann, Gregers G

2012-12-01

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 electroporation improves the cytotoxicity of mitomycin. In two cell lines, T24 (bladder cancer cell line) and DC3F (Chinese hamster fibroblast), exposure to different concentrations of mitomycin (0.01-2000μM) was tested with and without electroporation (6 pulses of 1kV/cm, duration: 99μs, frequency: 1Hz). Cell viability was assessed by colorimetric assay (MTT). For both cell lines, mitomycin's IC_50 was approximately 1000μM in both pulsed and unpulsed cells. On T24 cells, electroporation and mitomycin caused (relative reduction) RR of survival of: 25%, 31% and 29%, by concentrations 0μM, 500μM and 1000μM respectively. For DC3F cells, the RRs of survival were: 28%, 29%, and 33%, by concentrations 0μM, 500μM and 1000μM respectively. In conclusion, electroporation and mitomycin together are about 30% more effective than mitomycin alone. The results help to elucidate the additive effect of mitomycin and electric pulses and support the use of this combination in the treatment of bladder cancer. Copyright © 2012 Elsevier B.V. All rights reserved.

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

PubMed

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.

Rescue of photoreceptors by BDNF gene transfer using in vivo electroporation in the RCS rat of retinitis pigmentosa.

PubMed

Zhang, Meng; Mo, Xiaofen; Fang, Yuan; Guo, Wenyi; Wu, Jihong; Zhang, Shenghai; Huang, Qian

2009-09-01

To investigate the feasibility of introducing brain-derived neurotrophic factor (BDNF) gene into retinal pigment epithelial cells in vivo by electroporation and whether this method can rescue photoreceptors of retinitis pigmentosa in Royal College Surgeons (RCS) rats. The BDNF-GFP fusion eukaryotic-expressing plasmid was constructed and subretinally or intravitreously injected into the eyes of RCS rats followed by in vivo electroporation. The expression of BDNF mRNA and protein was detected by RT-PCR and Western immunoblot analysis. The number of surviving photoreceptors was counted, and the TdT-dUTP terminal nick-end labeling (TUNEL) method was used to detect the apoptotic retinal cells at different timepoints after introduction of BDNF plasmid. Treated eyes showed a significantly higher rescue ratio and a lower number of TUNEL-positive photoreceptors than did the control eyes at various timepoints. These findings provide evidence that electroporation is an effective method for gene transfer into retinal pigment epithelial cells, and the rescue of photoreceptors can be achieved by BDNF gene transfection with electroporation.

[The electroporation effects of high power pulse microwave and electromagnetic pulse irradiation on the membranes of cardiomyocyte cells and the mechanism therein involved].

PubMed

Deng, Hua; Wang, Dewen; Peng, Ruiyun; Wang, Shuiming; Chen, Jiankui; Zhang, Sa; Dong, Bo; Wang, Xiaomin

2005-08-01

Though there is ongoing public concern on potential hazards and risk of electromagnetic radiation, the bioeffects mechanism of electromagnetic fields remains obscure. Heart is one of the organs susceptive to electromagnetic fields (EMF). This study was designed to assess the influence of high power pulse microwave and electromagnetic pulse irradiation on cardiomyocytes, to explore the critical mechanism of electromagnetic fields, and to explain the regular course of injury caused by exposure to pulse EMF. Cultured cardiomyocytes were irradiated by high power pulse microwave and electromagnetic pulse first, then a series of apparatus including atom force microscope, laser scanning confocal microscope and flow cytometer were used to examine the changes of cell membrane conformation, structure and function. After irradiation, the cardiomyocytes pulsated slower or stop, the cells conformation was abnormal, the cells viability declined, and the percentage of apoptosis and necrosis increased significantly (P< 0.01). The cell membrane had pores unequal in size, and lost its penetration character. The concentration of Na+, K+, Ca2+, Cl-, Mg2+, Ca2+ and P3+ in cell culture medium increased significantly (P< 0.01). and the concentration of Ca2+ in cells ([Ca2+]i) decreased significantly (P<0.01). The results indicated that cardiomyocytes are susceptible to non-ionizing radiation. Pulse electromagnetic field can induce cardiomyocytes electroporation, and can do great damage to cells conformation, structure and function. Electroporation is one of the most critical mechanisms to explain the athermal effects of electromagnetic radiation.

Electroporation of a multivalent DNA vaccine cocktail elicits a protective immune response against anthrax and plague.

PubMed

Albrecht, Mark T; Livingston, Brian D; Pesce, John T; Bell, Matt G; Hannaman, Drew; Keane-Myers, Andrea M

2012-07-06

Electroporation of DNA vaccines represents a platform technology well positioned for the development of multivalent biodefense vaccines. To evaluate this hypothesis, three vaccine constructs were produced using codon-optimized genes encoding Bacillus anthracis Protective Antigen (PA), and the Yersinia pestis genes LcrV and F1, cloned into pVAX1. A/J mice were immunized on a prime-boost schedule with these constructs using the electroporation-based TriGrid Delivery System. Immunization with the individual pDNA vaccines elicited higher levels of antigen-specific IgG than when used in combination. DNA vaccine effectiveness was proven, the pVAX-PA titers were toxin neutralizing and fully protective against a lethal B. anthracis spore challenge when administered alone or co-formulated with the plague pDNA vaccines. LcrV and F1 pVAX vaccines against plague were synergistic, resulting in 100% survival, but less protective individually and when co-formulated with pVAX-PA. These DNA vaccine responses were Th1/Th2 balanced with high levels of IFN-γ and IL-4 in splenocyte recall assays, contrary to complimentary protein Alum vaccinations displaying a Th2 bias with increased IL-4 and low levels of IFN-γ. These results demonstrate the feasibility of electroporation to deliver and maintain the overall efficacy of an anthrax-plague DNA vaccine cocktail whose individual components have qualitative immunological differences when combined. Published by Elsevier Ltd.

A microfluidic flow-through device for high throughput electrical lysis of bacterial cells based on continuous dc voltage.

PubMed

Wang, Hsiang-Yu; Bhunia, Arun K; Lu, Chang

2006-12-15

Interest in electrical lysis of biological cells on a microfludic platform has increased because it allows for the rapid recovery of intracellular contents without introducing lytic agents. In this study we demonstrated a simple microfluidic flow-through device which lysed Escherichia coli cells under a continuous dc voltage. The E. coli cells had previously been modified to express green fluorescent protein (GFP). In our design, the cell lysis only happened in a defined section of a microfluidic channel due to the local field amplification by geometric modification. The geometric modification also effectively decreased the required voltage for lysis by several folds. We found that local field strength of 1000-1500 V/cm was required for nearly 100% cell death. This threshold field strength was considerably lower than the value reported in the literature, possibly due to the longer duration of the field [Lee, S.W., Tai, Y.C., 1999. Sens. Actuators A: Phys. 73, 74-79]. Cell lysis was detected by both plate count and fluorescence spectroscopy. The cell membrane was completely disintegrated in the lysis section of the microfluidic device, when the field strength was higher than 2000 V/cm. The devices were fabricated using low-cost soft lithography with channel widths considerably larger than the cell size to avoid clogging and ensure stable performance. Our tool will be ideal for high throughput processing of bacterial cells for chemical analysis of intracellular contents such as DNA and proteins. The application of continuous dc voltage greatly simplified the instrumentation compared to devices using electrical pulses for similar purposes. In principle, the same approach can also be applied for lysis of mammalian cells and electroporative transfection.

A Phase-1 Clinical Trial of a DNA Vaccine for Venezuelan Equine Encephalitis Delivered by Intramuscular or Intradermal Electroporation

DTIC Science & Technology

2016-05-25

A Phase 1 clinical trial of a DNA vaccine for Venezuelan equine encephalitis delivered by intramuscular or intradermal electroporation Drew... vaccines against VEEV available in the United States. We developed a candidate DNA vaccine expressing the E3-E2-6K-E1 genes of VEEV (pWRG/VEEV) and...groups and were vaccinated with high and low doses of pWRG/VEE or a saline placebo by intramuscular (IM) or intradermal (ID) electroporation (EP

Efficient mouse genome engineering by CRISPR-EZ technology.

PubMed

Modzelewski, Andrew J; Chen, Sean; Willis, Brandon J; Lloyd, K C Kent; Wood, Joshua A; He, Lin

2018-06-01

CRISPR/Cas9 technology has transformed mouse genome editing with unprecedented precision, efficiency, and ease; however, the current practice of microinjecting CRISPR reagents into pronuclear-stage embryos remains rate-limiting. We thus developed CRISPR ribonucleoprotein (RNP) electroporation of zygotes (CRISPR-EZ), an electroporation-based technology that outperforms pronuclear and cytoplasmic microinjection in efficiency, simplicity, cost, and throughput. In C57BL/6J and C57BL/6N mouse strains, CRISPR-EZ achieves 100% delivery of Cas9/single-guide RNA (sgRNA) RNPs, facilitating indel mutations (insertions or deletions), exon deletions, point mutations, and small insertions. In a side-by-side comparison in the high-throughput KnockOut Mouse Project (KOMP) pipeline, CRISPR-EZ consistently outperformed microinjection. Here, we provide an optimized protocol covering sgRNA synthesis, embryo collection, RNP electroporation, mouse generation, and genotyping strategies. Using CRISPR-EZ, a graduate-level researcher with basic embryo-manipulation skills can obtain genetically modified mice in 6 weeks. Altogether, CRISPR-EZ is a simple, economic, efficient, and high-throughput technology that is potentially applicable to other mammalian species.

Plasmid DNA vaccination using skin electroporation promotes poly-functional CD4 T-cell responses.

PubMed

Bråve, Andreas; Nyström, Sanna; Roos, Anna-Karin; Applequist, Steven E

2011-03-01

Plasmid DNA vaccination using skin electroporation (EP) is a promising method able to elicit robust humoral and CD8(+) T-cell immune responses while limiting invasiveness of delivery. However, there is still only limited data available on the induction of CD4(+) T-cell immunity using this method. Here, we compare the ability of homologous prime/boost DNA vaccinations by skin EP and intramuscular (i.m.) injection to elicit immune responses by cytokine enzyme-linked immunosorbent spot (ELISPOT) assay, as well as study the complexity of CD4(+) T-cell responses to the human immunodeficiency virus antigen Gag, using multiparamater flow cytometry. We find that DNA vaccinations by skin EP and i.m. injection are capable of eliciting both single- and poly-functional vaccine-specific CD4(+) T cells. However, although DNA delivered by skin EP was administered at a five-fold lower dose it elicited significant increases in the magnitude of multiple-cytokine producers compared with i.m. immunization suggesting that the skin EP could provide greater poly-functional T-cell help, a feature associated with successful immune defense against infectious agents.

Histological and Finite Element Analysis of Cell Death due to Irreversible Electroporation

PubMed Central

Long, G.; Bakos, G.; Shires, P. K.; Gritter, L.; Crissman, J. W.; Harris, J. L.; Clymer, J. W.

2014-01-01

Irreversible electroporation (IRE) has been shown to be an effective method of killing cells locally. In contrast to radiofrequency ablation, the mechanism by which cells are thought to die via IRE is the creation of pores in cell membranes, without substantial increase in tissue temperature. To determine the degree to which cell death is non-thermal, we evaluated IRE in porcine hepatocytes in vivo. Using pulse widths of 10μs, bursts of 3 kV square-wave pulses were applied through a custom probe to the liver of an anesthetized pig. Affected tissue was evaluated histologically via stainings of hematoxylin & eosin (H&E), nitroblue tetrazolium (NBT) to monitor cell respiration and TUNEL to gauge apoptosis. Temperature was measured during the application of electroporation, and heat transfer was modeled via finite element analysis. Cell death was calculated via Arrhenius kinetics. Four distinct zones were observed within the ring return electrode; heat-fixed tissue, coagulation, necrotic, and viable. The Arrhenius damage integral estimated complete cell death only in the first zone, where the temperature exceeded 70°C, and partial or no cell death in the other zones, where maximum temperature was approximately 45°C. Except for a limited area near the electrode tip, cell death in IRE is predominantly due to a non-thermal mechanism. PMID:24000980

Case report: Irreversible electroporation for locally advanced pancreatic cancer.

PubMed

Orcutt, Sonia; Kis, Bela; Malafa, Mokenge

2017-01-01

For patients with pancreatic adenocarcinoma who are not candidates for surgical resection, long-term survival is poor, even with currently available systemic and radiation therapy options. However, for those with locally advanced disease who do not have distant metastasis, locoregional control of the tumor has the potential to improve long-term outcomes. A newly developed technology, irreversible electroporation, has advantages over traditional thermal ablation with unresectable cancers in this location. In our case report, we describe the first patient treated with irreversible electroporation at our institution for locally advanced pancreatic cancer. The patient is a 63-year-old man who had a partial response to standard chemotherapy and radiation, but was found on operative assessment to have persistently unresectable disease. He therefore underwent irreversible electroporation to the pancreatic mass. His postoperative course was complicated by delayed gastric emptying and wound infection. Three months after surgery, he had no evidence of distant or recurrent disease. Irreversible electroporation for locally advanced pancreatic cancer is an emerging technique which attempts to improve local control of locally advanced, non-metastatic pancreatic cancer. Early data have demonstrated the potential for improved long-term survival in these patients, although further studies are needed to confirm safety and efficacy of this technique. While there is a positive outlook for the use of irreversible electroporation for locally advanced pancreas cancer, there remain some uncertainties surrounding this therapy, which underscores the importance of future research in this area. Copyright © 2017. Published by Elsevier Ltd.

Microelectronic electroporation array

NASA Astrophysics Data System (ADS)

Johnson, Lee J.; Shaffer, Kara J.; Skeath, Perry; Perkins, Frank K.; Pancrazio, Joseph; Scribner, Dean

2004-06-01

Gene Array technology has allowed for the study of gene binding by creating thousands of potential binding sites on a single device. A limitation of the current technology is that the effects of the gene and the gene-derived proteins cannot be studied in situ the same way, thousand site cell arrays are not readily available. We propose a new device structure to study the effects of gene modification on cells. This new array technology uses electroporation to target specific areas within a cell culture for transfection of genes. Electroporation arrays will allow high throughput analysis of gene effects on a given cell's response to a stress or a genes ability to restore normal cell function in disease modeling cells. Fluorescent imaging of dye labeled indicator molecules or cell viability will provide results indicating the most effective genes. The electroporation array consists of a microelectronic circuit, ancillary electronics, protecting electrode surface for cell culturing and a perfusion system for gene or drug delivery. The advantages of the current device are that there are 3200 sites for electroporation, all or any subsets of the electrodes can be activated. The cells are held in place by the electrode material. This technology could also be applied to high throughput screening of cell impermeant drugs.

Scalable Device for Automated Microbial Electroporation in a Digital Microfluidic Platform.

PubMed

Madison, Andrew C; Royal, Matthew W; Vigneault, Frederic; Chen, Liji; Griffin, Peter B; Horowitz, Mark; Church, George M; Fair, Richard B

2017-09-15

Electrowetting-on-dielectric (EWD) digital microfluidic laboratory-on-a-chip platforms demonstrate excellent performance in automating labor-intensive protocols. When coupled with an on-chip electroporation capability, these systems hold promise for streamlining cumbersome processes such as multiplex automated genome engineering (MAGE). We integrated a single Ti:Au electroporation electrode into an otherwise standard parallel-plate EWD geometry to enable high-efficiency transformation of Escherichia coli with reporter plasmid DNA in a 200 nL droplet. Test devices exhibited robust operation with more than 10 transformation experiments performed per device without cross-contamination or failure. Despite intrinsic electric-field nonuniformity present in the EP/EWD device, the peak on-chip transformation efficiency was measured to be 8.6 ± 1.0 × 10 8 cfu·μg -1 for an average applied electric field strength of 2.25 ± 0.50 kV·mm -1 . Cell survival and transformation fractions at this electroporation pulse strength were found to be 1.5 ± 0.3 and 2.3 ± 0.1%, respectively. Our work expands the EWD toolkit to include on-chip microbial electroporation and opens the possibility of scaling advanced genome engineering methods, like MAGE, into the submicroliter regime.

Modification of Schwann Cell Gene Expression by Electroporation in vivo

PubMed Central

Aspalter, Manuela; Vyas, Alka; Feiner, Jeffrey; Griffin, John; Brushart, Thomas; Redett, Richard

2009-01-01

Clinical outcomes of nerve grafting are often inferior to those of end-to-end nerve repair. This may be due, in part, to the routine use of cutaneous nerve to support motor axon regeneration. In previous work, we have demonstrated that Schwann cells express distinct sensory and motor phenotypes, and that these promote regeneration in a modality-specific fashion. Intra-operative modification of graft Schwann cell phenotype might therefore improve clinical outcomes. This paper demonstrates the feasibility of electroporating genes into intact nerve to modify Schwann cell gene expression. Initial trials established 70 V, 5 ms as optimum electroporation parameters. Intact, denervated, and reinnervated rat tibial nerves were electroporated with the YFP gene and evaluated serially by counting S-100 positive cells that expressed YFP. In intact nerve, a mean of 28% of Schwann cells expressed the gene at 3 days, falling to 20% at 7 days with little expression at later times. There were no significant differences among the three groups at each time period. Electronmicroscopic evaluation of treated, intact nerve revealed only occasional demyelination and axon degeneration. Intraoperative electroporation of nerve graft is thus a practical means of altering Schwann cell gene expression without the risks inherent in viral transfection. PMID:18834904

Theory and in vivo application of electroporative gene delivery.

PubMed

Somiari, S; Glasspool-Malone, J; Drabick, J J; Gilbert, R A; Heller, R; Jaroszeski, M J; Malone, R W

2000-09-01

Efficient and safe methods for delivering exogenous genetic material into tissues must be developed before the clinical potential of gene therapy will be realized. Recently, in vivo electroporation has emerged as a leading technology for developing nonviral gene therapies and nucleic acid vaccines (NAV). Electroporation (EP) involves the application of pulsed electric fields to cells to enhance cell permeability, resulting in exogenous polynucleotide transit across the cytoplasmic membrane. Similar pulsed electrical field treatments are employed in a wide range of biotechnological processes including in vitro EP, hybridoma production, development of transgenic animals, and clinical electrochemotherapy. Electroporative gene delivery studies benefit from well-developed literature that may be used to guide experimental design and interpretation. Both theory and experimental analysis predict that the critical parameters governing EP efficacy include cell size and field strength, duration, frequency, and total number of applied pulses. These parameters must be optimized for each tissue in order to maximize gene delivery while minimizing irreversible cell damage. By providing an overview of the theory and practice of electroporative gene transfer, this review intends to aid researchers that wish to employ the method for preclinical and translational gene therapy, NAV, and functional genomic research.

Glass Bead-based Genetic Transformation:An Efficient Method for Transformation of Thraustochytrid Microorganisms.

PubMed

Adachi, Takumi; Sahara, Takehiko; Okuyama, Hidetoshi; Morita, Naoki

2017-07-01

Here, we describe a new method for genetic transformation of thraustochytrids, well-known producers of polyunsaturated fatty acids (PUFAs) like docosahexaenoic acid, by combining mild glass (zirconia) bead treatment and electroporation. Because the cell wall is a barrier against transfer of exogenous DNA into cells, gentle vortexing of cells with glass beads was performed prior to electroporation for partial cell wall disruption. G418-resistant transformants of thraustochytrid cells (Aurantiochytrium limacinum strain SR21 and thraustochytrid strain 12B) were successfully obtained with good reproducibility. The method reported here is simpler than methods using enzymes to generate spheroplasts and may provide advantages for PUFA production by using genetically modified thraustochytrids.

Recovery of viral RNA and infectious foot-and-mouth disease virus from positive lateral-flow devices.

PubMed

Fowler, Veronica L; Bankowski, Bartlomiej M; Armson, Bryony; Di Nardo, Antonello; Valdazo-Gonzalez, Begoña; Reid, Scott M; Barnett, Paul V; Wadsworth, Jemma; Ferris, Nigel P; Mioulet, Valérie; King, Donald P

2014-01-01

Foot-and-mouth disease Virus (FMDV) is an economically important, highly contagious picornavirus that affects both wild and domesticated cloven hooved animals. In developing countries, the effective laboratory diagnosis of foot-and-mouth disease (FMD) is often hindered by inadequate sample preservation due to difficulties in the transportation and storage of clinical material. These factors can compromise the ability to detect and characterise FMD virus in countries where the disease is endemic. Furthermore, the high cost of sending infectious virus material and the biosecurity risk it presents emphasises the need for a thermo-stable, non-infectious mode of transporting diagnostic samples. This paper investigates the potential of using FMDV lateral-flow devices (LFDs) for dry transportation of clinical samples for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by electroporation. FMDV positive samples (epithelial suspensions and cell culture isolates) representing four FMDV serotypes were applied to antigen LFDs: after which it was possible to recover viral RNA that could be detected using real-time RT-PCR. Using this nucleic acid, it was also possible to recover VP1 sequences and also successfully utilise protocols for amplification of complete FMD virus genomes. It was not possible to recover infectious FMDV directly from the LFDs, however following electroporation into BHK-21 cells and subsequent cell passage, infectious virus could be recovered. Therefore, these results support the use of the antigen LFD for the dry, non-hazardous transportation of samples from FMD endemic countries to international reference laboratories.

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

PubMed Central

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

Cryopreservation of Human Adipose-Derived Stem Cells in Combination with Trehalose and Reversible Electroporation.

PubMed

Dovgan, Barbara; Barlič, Ariana; Knežević, Miomir; Miklavčič, Damijan

2017-02-01

New cryopreservation approaches for medically applicable cells are of great importance in clinical medicine. Current protocols employ the use of dimethyl sulfoxide (DMSO), which is toxic to cells and causes undesirable side effects in patients, such as cardiac arrhythmias, neurological events, and others. Trehalose, a nontoxic disaccharide, has been already studied as a cryoprotectant. However, an efficient approach for loading this impermeable sugar into mammalian cells is missing. In our study, we assessed the efficiency of combining reversible electroporation and trehalose for cryopreservation of human adipose-derived stem cells. First, we determined reversible electroporation threshold by loading of propidium iodide into cells. The highest permeabilization while maintaining high cell viability was reached at 1.5 kV/cm, at 8 pulses, 100 µs, and 1 Hz. Second, cells were incubated in 250 or 400 mM trehalose and electroporated before cryopreservation. After thawing, 83.8 ± 1.8 % (mean ± SE) cell recovery was obtained at 250 mM trehalose. By using a standard freezing protocol (10 % DMSO in 90 % fetal bovine serum), cell survival after thawing was about 91.5 ± 1.6 %. We also evaluated possible effects of electroporation on cells' functionality before and after thawing. Successful cell growth and efficient adipogenic and osteogenic differentiation were achieved. In conclusion, electroporation seems to be an efficient method for loading nonpermeable trehalose into human adipose-derived stem cells, allowing long-term cryopreservation in DMSO-free and xeno-free conditions.

Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor.

PubMed

Zhao, Yangbing; Moon, Edmund; Carpenito, Carmine; Paulos, Chrystal M; Liu, Xiaojun; Brennan, Andrea L; Chew, Anne; Carroll, Richard G; Scholler, John; Levine, Bruce L; Albelda, Steven M; June, Carl H

2010-11-15

Redirecting T lymphocyte antigen specificity by gene transfer can provide large numbers of tumor-reactive T lymphocytes for adoptive immunotherapy. However, safety concerns associated with viral vector production have limited clinical application of T cells expressing chimeric antigen receptors (CAR). T lymphocytes can be gene modified by RNA electroporation without integration-associated safety concerns. To establish a safe platform for adoptive immunotherapy, we first optimized the vector backbone for RNA in vitro transcription to achieve high-level transgene expression. CAR expression and function of RNA-electroporated T cells could be detected up to a week after electroporation. Multiple injections of RNA CAR-electroporated T cells mediated regression of large vascularized flank mesothelioma tumors in NOD/scid/γc(-/-) mice. Dramatic tumor reduction also occurred when the preexisting intraperitoneal human-derived tumors, which had been growing in vivo for >50 days, were treated by multiple injections of autologous human T cells electroporated with anti-mesothelin CAR mRNA. This is the first report using matched patient tumor and lymphocytes showing that autologous T cells from cancer patients can be engineered to provide an effective therapy for a disseminated tumor in a robust preclinical model. Multiple injections of RNA-engineered T cells are a novel approach for adoptive cell transfer, providing flexible platform for the treatment of cancer that may complement the use of retroviral and lentiviral engineered T cells. This approach may increase the therapeutic index of T cells engineered to express powerful activation domains without the associated safety concerns of integrating viral vectors. Copyright © 2010 AACR.

Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor

PubMed Central

Zhao, Yangbing; Moon, Edmund; Carpenito, Carmine; Paulos, Chrystal M.; Liu, Xiaojun; Brennan, Andrea L; Chew, Anne; Carroll, Richard G.; Scholler, John; Levine, Bruce L.; Albelda, Steven M.; June, Carl H.

2010-01-01

Redirecting T lymphocyte antigen specificity by gene transfer can provide large numbers of tumor reactive T lymphocytes for adoptive immunotherapy. However, safety concerns associated with viral vector production have limited clinical application of T cells expressing chimeric antigen receptors (CARs). T lymphocytes can be gene modified by RNA electroporation without integration-associated safety concerns. To establish a safe platform for adoptive immunotherapy, we first optimized the vector backbone for RNA in vitro transcription to achieve high level transgene expression. CAR expression and function of RNA-electroporated T cells could be detected up to a week post electroporation. Multiple injections of RNA CAR electroporated T cells mediated regression of large vascularized flank mesothelioma tumors in NOD/scid/γc(−/−) mice. Dramatic tumor reduction also occurred when the pre-existing intraperitoneal human-derived tumors, that had been growing in vivo for over 50 days, were treated by multiple injections of autologous human T cells electroporated with anti-mesothelin CAR mRNA. This is the first report using matched patient tumor and lymphocytes demonstrating that autologous T cells from cancer patients can be engineered to provide an effective therapy for a disseminated tumor in a robust preclinical model. Multiple injections of RNA engineered T cells are a novel approach for adoptive cell transfer, providing flexible platform for the treatment of cancer that may complement the use of retroviral and lentiviral engineered T cells. This approach may increase the therapeutic index of T cells engineered to express powerful activation domains without the associated safety concerns of integrating viral vectors. PMID:20926399

Effect of tape stripping and adjuvants on immune response after intradermal DNA electroporation.

PubMed

Vandermeulen, Gaëlle; Daugimont, Liévin; Richiardi, Hervé; Vanderhaeghen, Marie-Lise; Lecouturier, Nathalie; Ucakar, Bernard; Préat, Véronique

2009-07-01

DNA vaccines require both efficient delivery methods and appropriate adjuvants. Based on their mechanisms of action, we hypothesised that some adjuvants could enhance vaccine immunogenicity or direct the response towards Th1 profile after intradermal DNA electroporation. After intradermal electroporation of plasmid DNA encoding luciferase, mice received hyaluronidase, imiquimod, monophosphoryl lipid A or were tape stripped in order to modulate the immune response against the encoded protein. We measured total immunoglobulin G, IgG1, IgG2a titres and the cytokines produced by splenocyte cultures to assess both humoral and cellular response. The effect of tape stripping on the response against intradermally delivered ovalbumin protein was also assessed. Neither hyaluronidase nor imiquimod improved the immune response against the encoded luciferase. Monophosphoryl lipid A did not modify the cytokines production but increased the anti-luciferase IgG2a titres. Tape stripping significantly increased anti-luciferase IgG2a and IFN-gamma responses. It also enhanced the humoral response after intradermal injection of the ovalbumin protein. Tape stripping is able to increase the Th1 immune response against both DNA and protein vaccines. Therefore, tape stripping appears to have interesting adjuvant effect on intradermal vaccination.

Why we should not routinely apply irreversible electroporation as an alternative curative treatment modality for localized prostate cancer at this stage.

PubMed

Wendler, J J; Ganzer, R; Hadaschik, B; Blana, A; Henkel, T; Köhrmann, K U; Machtens, S; Roosen, A; Salomon, G; Sentker, L; Witzsch, U; Schlemmer, H P; Baumunk, D; Köllermann, J; Schostak, M; Liehr, U B

2017-01-01

Irreversible electroporation (IRE), a new tissue ablation procedure available since 2007, could meet the requirements for ideal focal therapy of prostate cancer with its postulated features, especially the absence of a thermal ablation effect. Thus far, there is not enough evidence of its effectiveness or adverse effects to justify its use as a definitive treatment option for localized prostate cancer. Moreover, neither optimal nor individual treatment parameters nor uniform endpoints have been defined thus far. No advantages over established treatment procedures have as yet been demonstrated. Nevertheless, IRE is now being increasingly applied for primary prostate cancer therapy outside clinical trials, not least through active advertising in the lay press. This review reflects the previous relevant literature on IRE of the prostate or prostate cancer and shows why we should not adopt IRE as a routine treatment modality at this stage.

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

PubMed

Yao, Chenguo; Lv, Yanpeng; Dong, Shoulong; Zhao, Yajun; Liu, Hongmei

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.

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

PubMed Central

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

The effects of different concentrations of ccBA-GFP promoter with electroporation methods on the quality of koi sperm (Cyprinus carpio var. koi)

NASA Astrophysics Data System (ADS)

Soeprijanto, A.; Aisyah, D.

2018-04-01

The effectiveness of the use of promoter concentration which will be inserted into the Koi sperm as the medium of gene transfer is important. The objective of this research is to find out the influence of the adding of different concentrations of the ccBA-GFP promoter with electroporation methods to the motility, viability and the fertilization rate of the Koi sperm. This study was conducted at Central Lab of Life Sciences Brawijaya University in April 2017. Electroporation methods were conducted by using 30-volt voltage, 4 times shocks with 0.5 seconds per shock. The treatment of different concentration was done through 3 types of ccBA-GFP promoter concentration, namely: 10 ng/µl, 30 ng/µl, and 50 ng/µl. The best motility percentage with the score of 4 is at the treatment A (10ng/µl concentration), the best viability percentage is 77.83 % at the treatment A (10 ng/µl concentration) and the best fertilization rate is 73.09 % at the treatment A (10 ng/µl concentration). The result shows that there is a relationship between the treatment given to the motility and viability of the Koi sperm, at which, the higher the shocks, the lower the percentage of the motility and viability of the Koi sperm.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

PubMed Central

Oudin, Madeleine Julie; Doherty, Patrick; Lalli, Giovanna

2013-01-01

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration. In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair. PMID:24326479

The photodynamic effect of far-red range phthalocyanines (AlPc and Pc green) supported by electropermeabilization in human gastric adenocarcinoma cells of sensitive and resistant type.

PubMed

Zielichowska, Anna; Saczko, Jolanta; Garbiec, Arnold; Dubińska-Magiera, Magda; Rossowska, Joanna; Surowiak, Paweł; Choromańska, Anna; Daczewska, Małgorzata; Kulbacka, Julita; Lage, Hermann

2015-02-01

Electroporation (EP) is commonly applied for effective drug transport thorough cell membranes based on the application of electromagnetic field. When applied with cytostatics, it is called electrochemotherapy (ECT) - a quite new method of cancer treatment. A high-voltage pulse causes the formation of temporary pores in the cell membrane which create an additional way for the intracellular drug transport. In the current work, EP was effectively merged with the already known photodynamic therapy (PDT) to selective photosensitizers' delivery to diseased tissue. The application of electroporation can reduce the dose of applied drug. The aim of research was to evaluate the effectiveness of photodynamic reaction using two near infrared cyanines (AlPc and Pc green) combined with electroporation in two human gastric adenocarcinoma cell lines. Two human cell lines - EPG85-257P (parental) and EPG85-257RDB (resistant to daunorubicin) - of gastric cancer were used. The effect of two photosensitizers (aluminum 1,8,15,22-tetrakis(-phenylthio)-29H,31H-phthalocyanine chloride and Phthalocyanine green) was investigated. The efficiency of EP parameters was assessed by propidium iodide uptake. The viability assay was applied to analyse EP, PDT and EP-PDT effect. Cyanine localization was determined by confocal microscopy. Immunocytochemical evaluation of manganese superoxide dismutase and glutathione S-transferase-pi was determined after applied therapies. PDT in combination with EP affected the viability of EPG85-257P and EPG85-257RDB cells negatively while both cyanine were used. The most evident changes were observed in the following concentrations: 15, 10 and 5μM. The optimal field strength for enhanced EP-PDT was 800 and 1200V/cm. AlPc distributed selectively in the lysosomes of parental cell line. PDT, enhanced by EP, caused decreased viability when compared to the application of PDT alone. Both phthalocyanines found to be more effective after electroporation. Due to the low concentration of light-sensitive compounds and safety of electroporation itself, a treatment plan can be an alternative therapeutic modality against gastric adenocarcinomas. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Targeted gene delivery in the cricket brain, using in vivo electroporation.

PubMed

Matsumoto, Chihiro Sato; Shidara, Hisashi; Matsuda, Koji; Nakamura, Taro; Mito, Taro; Matsumoto, Yukihisa; Oka, Kotaro; Ogawa, Hiroto

2013-12-01

The cricket (Gryllus bimaculatus) is a hemimetabolous insect that is emerging as a model organism for the study of neural and molecular mechanisms of behavioral traits. However, research strategies have been limited by a lack of genetic manipulation techniques that target the nervous system of the cricket. The development of a new method for efficient gene delivery into cricket brains, using in vivo electroporation, is described here. Plasmid DNA, which contained an enhanced green fluorescent protein (eGFP) gene, under the control of a G. bimaculatus actin (Gb'-act) promoter, was injected into adult cricket brains. Injection was followed by electroporation at a sufficient voltage. Expression of eGFP was observed within the brain tissue. Localized gene expression, targeted to specific regions of the brain, was also achieved using a combination of local DNA injection and fine arrangement of the electroporation electrodes. Further studies using this technique will lead to a better understanding of the neural and molecular mechanisms that underlie cricket behaviors. Copyright © 2013 Elsevier Ltd. All rights reserved.

Isolation of Lightning-Competent Soil Bacteria

PubMed Central

Cérémonie, Hélène; Buret, François; Simonet, Pascal; Vogel, Timothy M.

2004-01-01

Artificial transformation is typically performed in the laboratory by using either a chemical (CaCl2) or an electrical (electroporation) method. However, laboratory-scale lightning has been shown recently to electrotransform Escherichia coli strain DH10B in soil. In this paper, we report on the isolation of two “lightning-competent” soil bacteria after direct electroporation of the Nycodenz bacterial ring extracted from prairie soil in the presence of the pBHCRec plasmid (Tcr, Spr, Smr). The electrotransformability of the isolated bacteria was measured both in vitro (by electroporation cuvette) and in situ (by lightning in soil microcosm) and then compared to those of E. coli DH10B and Pseudomonas fluorescens C7R12. The electrotransformation frequencies measured reached 10−3 to 10−4 by electroporation and 10−4 to 10−5 by simulated lightning, while no transformation was observed in the absence of electrical current. Two of the isolated lightning-competent soil bacteria were identified as Pseudomonas sp. strains. PMID:15466589

A new way to generate cytolytic tumor-specific T cells: electroporation of RNA coding for a T cell receptor into T lymphocytes.

PubMed

Schaft, Niels; Dörrie, Jan; Müller, Ina; Beck, Verena; Baumann, Stefanie; Schunder, Tanja; Kämpgen, Eckhart; Schuler, Gerold

2006-09-01

Effective T cell receptor (TCR) transfer until now required stable retroviral transduction. However, retroviral transduction poses the threat of irreversible genetic manipulation of autologous cells. We, therefore, used optimized RNA transfection for transient manipulation. The transfection efficiency, using EGFP RNA, was >90%. The electroporation of primary T cells, isolated from blood, with TCR-coding RNA resulted in functional cytotoxic T lymphocytes (CTLs) (>60% killing at an effector to target ratio of 20:1) with the same HLA-A2/gp100-specificity as the parental CTL clone. The TCR-transfected T cells specifically recognized peptide-pulsed T2 cells, or dendritic cells electroporated with gp100-coding RNA, in an IFNgamma-secretion assay and retained this ability, even after cryopreservation, over 3 days. Most importantly, we show here for the first time that the electroporated T cells also displayed cytotoxicity, and specifically lysed peptide-loaded T2 cells and HLA-A2+/gp100+ melanoma cells over a period of at least 72 h. Peptide-titration studies showed that the lytic efficiency of the RNA-transfected T cells was similar to that of retrovirally transduced T cells, and approximated that of the parental CTL clone. Functional TCR transfer by RNA electroporation is now possible without the disadvantages of retroviral transduction, and forms a new strategy for the immunotherapy of cancer.

Web-based tool for visualization of electric field distribution in deep-seated body structures and planning of electroporation-based treatments.

PubMed

Marčan, Marija; Pavliha, Denis; Kos, Bor; Forjanič, Tadeja; Miklavčič, Damijan

2015-01-01

Treatments based on electroporation are a new and promising approach to treating tumors, especially non-resectable ones. The success of the treatment is, however, heavily dependent on coverage of the entire tumor volume with a sufficiently high electric field. Ensuring complete coverage in the case of deep-seated tumors is not trivial and can in best way be ensured by patient-specific treatment planning. The basis of the treatment planning process consists of two complex tasks: medical image segmentation, and numerical modeling and optimization. In addition to previously developed segmentation algorithms for several tissues (human liver, hepatic vessels, bone tissue and canine brain) and the algorithms for numerical modeling and optimization of treatment parameters, we developed a web-based tool to facilitate the translation of the algorithms and their application in the clinic. The developed web-based tool automatically builds a 3D model of the target tissue from the medical images uploaded by the user and then uses this 3D model to optimize treatment parameters. The tool enables the user to validate the results of the automatic segmentation and make corrections if necessary before delivering the final treatment plan. Evaluation of the tool was performed by five independent experts from four different institutions. During the evaluation, we gathered data concerning user experience and measured performance times for different components of the tool. Both user reports and performance times show significant reduction in treatment-planning complexity and time-consumption from 1-2 days to a few hours. The presented web-based tool is intended to facilitate the treatment planning process and reduce the time needed for it. It is crucial for facilitating expansion of electroporation-based treatments in the clinic and ensuring reliable treatment for the patients. The additional value of the tool is the possibility of easy upgrade and integration of modules with new functionalities as they are developed.

Web-based tool for visualization of electric field distribution in deep-seated body structures and planning of electroporation-based treatments

PubMed Central

2015-01-01

Background Treatments based on electroporation are a new and promising approach to treating tumors, especially non-resectable ones. The success of the treatment is, however, heavily dependent on coverage of the entire tumor volume with a sufficiently high electric field. Ensuring complete coverage in the case of deep-seated tumors is not trivial and can in best way be ensured by patient-specific treatment planning. The basis of the treatment planning process consists of two complex tasks: medical image segmentation, and numerical modeling and optimization. Methods In addition to previously developed segmentation algorithms for several tissues (human liver, hepatic vessels, bone tissue and canine brain) and the algorithms for numerical modeling and optimization of treatment parameters, we developed a web-based tool to facilitate the translation of the algorithms and their application in the clinic. The developed web-based tool automatically builds a 3D model of the target tissue from the medical images uploaded by the user and then uses this 3D model to optimize treatment parameters. The tool enables the user to validate the results of the automatic segmentation and make corrections if necessary before delivering the final treatment plan. Results Evaluation of the tool was performed by five independent experts from four different institutions. During the evaluation, we gathered data concerning user experience and measured performance times for different components of the tool. Both user reports and performance times show significant reduction in treatment-planning complexity and time-consumption from 1-2 days to a few hours. Conclusions The presented web-based tool is intended to facilitate the treatment planning process and reduce the time needed for it. It is crucial for facilitating expansion of electroporation-based treatments in the clinic and ensuring reliable treatment for the patients. The additional value of the tool is the possibility of easy upgrade and integration of modules with new functionalities as they are developed. PMID:26356007

Electroporation of Functional Bacterial Effectors into Mammalian Cells

DOE PAGES

Sontag, Ryan L.; Mihai, Cosmin; Orr, Galya; ...

2015-01-19

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

A highly efficient transposon mutagenesis system for the tomato pathogen Clavibacter michiganensis subsp. michiganensis.

PubMed

Kirchner, O; Gartemann, K H; Zellermann, E M; Eichenlaub, R; Burger, A

2001-11-01

A transposon mutagenesis system for Clavibacter michiganensis subsp. michiganensis was developed based on antibiotic resistance transposons that were derived from the insertion element IS1409 from Arthrobacter sp. strain TM1 NCIB12013. As a prerequisite, the electroporation efficiency was optimized by using unmethylated DNA and treatment of the cells with glycine such that about 5 x 10(6) transformants per microg of DNA were generally obtained. Electroporation of C. michiganensis subsp. michiganensis with a suicide vector carrying transposon Tn1409C resulted in approximately 1 x 10(3) transposon mutants per pg of DNA and thus is suitable for saturation mutagenesis. Analysis of Tn1409C insertion sites suggests a random mode of transposition. Transposition of Tn1409C was also demonstrated for other subspecies of C. michiganensis.

Rapid and efficient gene delivery into the adult mouse brain via focal electroporation

PubMed Central

Nomura, Tadashi; Nishimura, Yusuke; Gotoh, Hitoshi; Ono, Katsuhiko

2016-01-01

In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner. PMID:27430903

On-chip, multisite extracellular and intracellular recordings from primary cultured skeletal myotubes

PubMed Central

Rabieh, Noha; Ojovan, Silviya M.; Shmoel, Nava; Erez, Hadas; Maydan, Eilon; Spira, Micha E.

2016-01-01

In contrast to the extensive use of microelectrode array (MEA) technology in electrophysiological studies of cultured neurons and cardiac muscles, the vast field of skeletal muscle research has yet to adopt the technology. Here we demonstrate an empowering MEA technology for high quality, multisite, long-term electrophysiological recordings from cultured skeletal myotubes. Individual rat skeletal myotubes cultured on micrometer sized gold mushroom-shaped microelectrode (gMμE) based MEA tightly engulf the gMμEs, forming a high seal resistance between the myotubes and the gMμEs. As a consequence, spontaneous action potentials generated by the contracting myotubes are recorded as extracellular field potentials with amplitudes of up to 10 mV for over 14 days. Application of a 10 ms, 0.5–0.9 V voltage pulse through the gMμEs electroporated the myotube membrane, and transiently converted the extracellular to intracellular recording mode for 10–30 min. In a fraction of the cultures stable attenuated intracellular recordings were spontaneously produced. In these cases or after electroporation, subthreshold spontaneous potentials were also recorded. The introduction of the gMμE-MEA as a simple-to-use, high-quality electrophysiological tool together with the progress made in the use of cultured human myotubes opens up new venues for basic and clinical skeletal muscle research, preclinical drug screening, and personalized medicine. PMID:27812002

Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection

PubMed Central

Michlewski, Gracjan; Finnegan, David J.; Elfick, Alistair; Rosser, Susan J.

2017-01-01

Abstract Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing. PMID:28204586

Electroporation of the photosynthetic membrane: structural changes in protein and lipid-protein domains.

PubMed Central

Rosemberg, Y; Rotenberg, M; Korenstein, R

1994-01-01

A biological membrane undergoes a reversible permeability increase through structural changes in the lipid domain when exposed to high external electric fields. The present study shows the occurrence of electric field-induced changes in the conductance of the proton channel of the H(+)-ATPase as well as electric field-induced structural changes in the lipid-protein domain of photosystem (PS) II in the photosynthetic membrane. The study was carried out by analyzing the electric field-stimulated delayed luminescence (EPL), which originates from charge recombination in the protein complexes of PS I and II of photosynthetic vesicles. We established that a small fraction of the total electric field-induced conductance change was abolished by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the H(+)-ATPase. This reversible electric field-induced conductance change has characteristics of a small channel and possesses a lifetime < or = 1 ms. To detect electric field-induced changes in the lipid-protein domains of PS II, we examined the effects of phospholipase A2 (PLA2) on EPL. Higher values of EPL were observed from vesicles that were exposed in the presence of PLA2 to an electroporating electric field than to a nonelectroporating electric field. The effect of the electroporating field was a long-lived one, lasting for a period > or = 2 min. This effect was attributed to long-lived electric field-induced structural changes in the lipid-protein domains of PS II. PMID:7811916

Magnetic resonance imaging characteristics of nonthermal irreversible electroporation in vegetable tissue.

PubMed

Hjouj, Mohammad; Rubinsky, Boris

2010-07-01

We introduce and characterize the use of MRI for studying nonthermal irreversible electroporation (NTIRE) in a vegetative tissue model. NTIRE is a new minimally invasive surgical technique for tissue ablation in which microsecond, high electric-field pulses form nanoscale defects in the cell membrane that lead to cell death. Clinical NTIRE sequences were applied to a potato tuber tissue model. The potato is used for NTIRE studies because cell damage is readily visible with optical means through a natural oxidation process of released intracellular enzymes (polyphenol oxidase) and the formation of brown-black melanins. MRI sequences of the treated area were taken at various times before and after NTIRE and compared with photographic images. A comparison was made between T1W, T2W, FLAIR and STIR MRIs of NTIRE and photographic images. Some MRI sequences show changes in areas treated by irreversible electroporation. T1W and FLAIR produce brighter images of the treated areas. In contrast, the signal was lost from the treated area when a suppression technique, STIR, was used. There was similarity between optical photographic images of the treated tissue and MRIs of the same areas. This is the first study to characterize MRI of NTIRE in vegetative tissue. We find that NTIRE produces changes in vegetative tissue that can be imaged by certain MRI sequences. This could make MRI an effective tool to study the fundamentals of NTIRE in nonanimal tissue.

Efficient electroporation of DNA and protein into confluent and differentiated epithelial cells in culture.

PubMed

Deora, Ami A; Diaz, Fernando; Schreiner, Ryan; Rodriguez-Boulan, Enrique

2007-10-01

Electroporation-mediated delivery of molecules is a procedure widely used for transfecting complementary DNA in bacteria, mammalian and plant cells. This technique has proven very efficient for the introduction of macromolecules into cells in suspension culture and even into cells in their native tissue environment, e.g. retina and embryonic tissues. However, in spite of several attempts to date, there are no well-established procedures to electroporate polarized epithelial cells adhering to a tissue culture substrate (glass, plastic or filter). We report here the development of a simple procedure that uses available commercial equipment and works efficiently and reproducibly for a variety of epithelial cell lines in culture.

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

PubMed Central

Machado-Aranda, David; Raghavendran, Krishnan

2015-01-01

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. PMID:24510825

Recombineering Pseudomonas syringae

USDA-ARS?s Scientific Manuscript database

Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...

Influence of DMPS on the water retention capacity of electroporated stratum corneum: ATR-FTIR study.

PubMed

Sckolnick, Maria; Hui, Sek-Wen; Sen, Arindam

2008-02-28

Anionic lipids like phosphatidylserine are known to significantly enhance electroporation mediated transepidermal transport of polar solutes of molecular weights up to 10kDa. The underlying mechanism of the effect of anionic lipids on transdermal transport is not fully understood. The main barrier to transdermal transport lies within the intercellular lipid matrix (ILM) of the stratum corneum (SC) and our previous studies indicate that dimyristoyl phosphatidylserine (DMPS) can perturb the packing of this lipid matrix. Here we report on our investigation on water retention in the SC following electroporation in the presence and the absence of DMPS. The water content in the outer most layers of the SC of full thickness porcine skin was determined using ATR-FTIR-spectroscopy. The results show that in the presence of DMPS, the SC remains in a state of enhanced hydration for longer periods after electroporation. This increase in water retention in the SC by DMPS is likely to play an important role in trans-epidermal transport, since improved hydration of the skin barrier can be expected to increase the partitioning of polar solutes and possibly the permeability.

Alteration of gene expression by restriction enzymes electroporated into plant cells.

PubMed

Ashraf, M; Altschuler, M; Galasinski, S; Griffiths, T D

1993-06-01

The alteration in the expression of a beta-glucuronidase (GUS) reporter gene was used to monitor the effect of restriction endonucleases electroporated into the tobacco (Nicotiana tabacum L.) protoplasts. Restriction enzyme (RE) Hind III which does not have a recognition site within the gene cassette, had little effect on enzyme activity. In contrast restriction endonucleases Hae III and Sau3A1 which possess 8 and 16 recognition sites in the GUS cassette, were found to reduce the enzyme activity by 89% and 94% respectively when compared to control electroporations. Restriction-site mutation analysis (RSM) and Southern blot analysis indicated the enzymatic degradation of GUS coding sequence by the REs Hae III and Sau3A1. Results of this study suggest that on electroporation, REs can enter into plant cells and alter the expression of the GUS gene. The alteration of gene expression is thus correlated with the digestion of GUS template DNA. Future applications of this technique could include addressing fundamental questions with regard to DNA repair, site-specific recombination, identifying mutations, insertional mutagenesis, enhancement of stable transformation and gene tagging in plants.

Skin Electroporation: Effects on Transgene Expression, DNA Persistence and Local Tissue Environment

PubMed Central

Roos, Anna-Karin; Eriksson, Fredrik; Timmons, James A.; Gerhardt, Josefine; Nyman, Ulrika; Gudmundsdotter, Lindvi; Bråve, Andreas; Wahren, Britta; Pisa, Pavel

2009-01-01

Background Electrical pulses have been used to enhance uptake of molecules into living cells for decades. This technique, often referred to as electroporation, has become an increasingly popular method to enhance in vivo DNA delivery for both gene therapy applications as well as for delivery 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 and electroporation induced a significant up-regulation of pro-inflammatory genes. In vivo imaging of luciferase activity after electrovaccination demonstrated a rapid onset (minutes) and a long duration (months) of transgene expression. However, when the more immunogenic prostate specific antigen (PSA) was co-administered, PSA-specific T cells were induced and concurrently the luciferase expression became undetectable. Electroporation did not affect the long-term persistence of the PSA-expressing plasmid. Conclusions/Significance This study provides important insights to how DNA delivery by intradermal electrovaccination affects the local immunological responses of the skin, transgene expression and clearance of the plasmid. As the described vaccination approach is currently being evaluated in clinical trials, the data provided will be of high significance. PMID:19789652

piggyBac Transposon-Mediated Transgenesis in the Apicomplexan Parasite Eimeria tenella

PubMed Central

Su, Huali; Liu, Xianyong; Yan, Wenchao; Shi, Tuanyuan; Zhao, Xinxin; Blake, Damer P.; Tomley, Fiona M.; Suo, Xun

2012-01-01

piggyBac, a type II transposon that is useful for efficient transgenesis and insertional mutagenesis, has been used for effective and stable transfection in a wide variety of organisms. In this study we investigate the potential use of the piggyBac transposon system for forward genetics studies in the apicomplexan parasite Eimeria tenella. Using the restriction enzyme-mediated integration (REMI) method, E. tenella sporozoites were electroporated with a donor plasmid containing the enhanced yellow fluorescent protein (EYFP) gene flanked by piggyBac inverted terminal repeats (ITRs), an Asc I-linearized helper plasmid containing the transposase gene and the restriction enzyme Asc I. Subsequently, electroporated sporozoites were inoculated into chickens via the cloacal route and transfected progeny oocysts expressing EYFP were sorted by flow cytometry. A transgenic E. tenella population was selected by successive in vivo passage. Southern-blotting analysis showed that exogenous DNA containing the EYFP gene was integrated into the parasite genome at a limited number of integration sites and that the inserted part of the donor plasmid was the fragment located between the 5′ and 3′ ITRs as indicated by primer-specific PCR screening. Genome walking revealed that the insertion sites were TTAA-specific, which is consistent with the transposition characteristics of piggyBac. PMID:22768223

Immunogenicity and safety of xenogeneic vascular endothelial growth factor receptor-2 DNA vaccination in mice and dogs

PubMed Central

Denies, Sofie; Cicchelero, Laetitia; Polis, Ingeborgh; Sanders, Niek N.

2016-01-01

Vascular endothelial growth factor receptor-2 (VEGFR-2) is an attractive target in oncology due to its crucial role in angiogenesis. In this study a DNA vaccine coding for human VEGFR-2 was evaluated in healthy mice and dogs, administered by intradermal injection and electroporation. In mice, three doses and vaccination schedules were evaluated. Cellular immune responses were measured by intracellular IFN-gamma staining and a cytotoxicity assay and antibodies by ELISA. Safety was assessed by measuring regulatory T cells and myeloid derived suppressor cells and a wound healing assay. The vaccine was subsequently evaluated in dogs, which were vaccinated three times with 100μg. Cellular immune responses were measured by intracellular IFN-gamma staining and antibodies by a flow cytometric assay. In mice, maximal cellular responses were observed after two vaccinations with 5μg. Humoral responses continued to increase with higher dose and number of vaccinations. No abnormalities in the measured safety parameters were observed. The vaccine was also capable of eliciting a cellular and humoral immune response in dogs. No adverse effects were observed, but tolerability of the electroporation was poor. This study will facilitate the evaluation of the vaccine in tumor bearing animals, ranging from rodent models to dogs with spontaneous tumors. PMID:26871296

Dynamic effects and applications for nanosecond pulsed electric fields in cells and tissues

NASA Astrophysics Data System (ADS)

Beebe, Stephen J.; Blackmore, Peter F.; Hall, Emily; White, Jody A.; Willis, Lauren K.; Fauntleroy, Laura; Kolb, Juergen F.; Schoenbach, Karl H.

2005-04-01

Nanosecond, high intensity pulsed electric fields [nsPEFs] that are below the plasma membrane [PM] charging time constant have decreasing effects on the PM and increasing effects on intracellular structures and functions as the pulse duration decreases. When human cell suspensions were exposed to nsPEFs where the electric fields were sufficiently intense [10-300ns, <=300 kV/cm.], apoptosis signaling pathways could be activated in several cell models. Multiple apoptosis markers were observed in Jurkat, HL-60, 3T3L1-preadipocytes, and isolated rat adipocytes including decreased cell size and number, caspase activation, DNA fragmentation, and/or cytochrome c release into the cytoplasm. Phosphatidylserine externalization was observed as a biological response to nsPEFs in 3T3-L1 preadipocytes and p53-wildtype and -null human colon carcinoma cells. B10.2 mouse fibrosarcoma tumors that were exposed to nsPEFs ex vivo and in vivo exhibited DNA fragmentation, elevated caspase activity, and reduced size and weight compared to contralateral sham-treated control tumors. When nsPEF conditions were below thresholds for apoptosis and classical PM electroporation, non-apoptotic responses were observed similar to those initiated through PM purinergic receptors in HL-60 cells and thrombin in human platelets. These included Ca2+ mobilization from intracellular stores [endoplasmic reticulum] and subsequently through store-operated Ca2+ channels in the PM. In addition, platelet activation measured as aggregation responses were observed in human platelets. Finally, when nsPEF conditions followed classical electroporation-mediated transfection, the expression intensity and number of GFP-expressing cells were enhanced above cells exposed to electroporation conditions alone. These studies demonstrate that application of nsPEFs to cells or tissues can modulate cell-signaling mechanisms with possible applications as a new basic science tool, cancer treatment, wound healing, and gene therapy.

Recombineering using RecET from Pseudomonas syringae

USDA-ARS?s Scientific Manuscript database

Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...

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

PubMed Central

Takahashi, Shota; Asada, Atsushi; Matsuo, Minako; Kishikawa, Kenta; Mizuno, Akira

2015-01-01

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. PMID:26649904

Subretinal delivery and electroporation in pigmented and nonpigmented adult mouse eyes

PubMed Central

Nickerson, John M.; Goodman, Penny; Chrenek, Micah A.; Johnson, Christiana J.; Berglin, Lennart; Redmond, T. Michael.; Boatright, Jeffrey H.

2013-01-01

Subretinal injection offers one of the best ways to deliver many classes of drugs, reagents, cells and treatments to the photoreceptor, Müller, and retinal pigment epithelium (RPE) cells of the retina. Agents delivered to this space are placed within microns of the intended target cell, accumulating to high concentrations because there is no dilution due to transport processes or diffusion. Dilution in the interphotoreceptor space (IPS) is minimal because the IPS volume is only 10-20 microliters in the human eye and less than 1 microliter in the mouse eye. For gene delivery purposes, we wished to transfect the cells adjacent to the IPS in adult mouse eyes. Others transfect these cells in neonatal rats to study the development of the retina. In both neonates and adults, electroporation is found to be effective Here we describe the optimization of electroporation conditions for RPE cells in the adult mouse eye with naked plasmids. However, both techniques, subretinal injection and electroporation, present some technical challenges that require skill on the part of the surgeon to prevent untoward damage to the eye. Here we describe methods that we have used for the past ten years (1). PMID:22688698

Oral Mucosa Model for Electrochemotherapy Treatment of Dog Mouth Cancer: Ex Vivo, In Silico, and In Vivo Experiments.

PubMed

Suzuki, Daniela O H; Berkenbrock, José A; Frederico, Marisa J S; Silva, Fátima R M B; Rangel, Marcelo M M

2018-03-01

Electrochemotherapy (EQT) is a local cancer treatment well established to cutaneous and subcutaneous tumors. Electric fields are applied to biological tissue in order to improve membrane permeability for cytotoxic drugs. This phenomenon is called electroporation or electropermeabilization. Studies have reported that tissue conductivity is electric field dependent. Electroporation numerical models of biological tissues are essential in treatment planning. Tumors of the mouth are very common in dogs. Inadequate EQT treatment of oral tumor may be caused by significant anatomic variations between dogs and tumor position. Numerical models of oral mucosa and tumor allow the treatment planning and optimization of electrodes for each patient. In this work, oral mucosa conductivity during electroporation was characterized by measuring applied voltage and current of ex vivo rats. This electroporation model was used with a spontaneous canine oral melanoma. The model outcomes of oral tumor EQT is applied in different parts of the oral cavity including near bones and the hard palate. The numerical modeling for treatment planning will help the development of new electrodes and increase the EQT effectiveness. © 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

The systematic study of the electroporation and electrofusion of B16-F1 and CHO cells in isotonic and hypotonic buffer.

PubMed

Usaj, Marko; Kanduser, Masa

2012-09-01

The fusogenic state of the cell membrane can be induced by external electric field. When two fusogenic membranes are in close contact, cell fusion takes place. An appropriate hypotonic treatment of cells before the application of electric pulses significantly improves electrofusion efficiency. How hypotonic treatment improves electrofusion is still not known in detail. Our results indicate that at given induced transmembrane potential electroporation was not affected by buffer osmolarity. In contrast to electroporation, cells' response to hypotonic treatment significantly affects their electrofusion. High fusion yield was observed when B16-F1 cells were used; this cell line in hypotonic buffer resulted in 41 ± 9 % yield, while in isotonic buffer 32 ± 11 % yield was observed. Based on our knowledge, these fusion yields determined in situ by dual-color fluorescence microscopy are among the highest in electrofusion research field. The use of hypotonic buffer was more crucial for electrofusion of CHO cells; the fusion yield increased from below 1 % in isotonic buffer to 10 ± 4 % in hypotonic buffer. Since the same degree of cell permeabilization was achieved in both buffers, these results indicate that hypotonic treatment significantly improves fusion yield. The effect could be attributed to improved physical contact of cell membranes or to enhanced fusogenic state of the cell membrane itself.

A high efficiency gene disruption strategy using a positive-negative split selection marker and electroporation for Fusarium oxysporum.

PubMed

Liang, Liqin; Li, Jianqiang; Cheng, Lin; Ling, Jian; Luo, Zhongqin; Bai, Miao; Xie, Bingyan

2014-11-01

The Fusarium oxysporum species complex consists of fungal pathogens that cause serial vascular wilt disease on more than 100 cultivated species throughout the world. Gene function analysis is rapidly becoming more and more important as the whole-genome sequences of various F. oxysporum strains are being completed. Gene-disruption techniques are a common molecular tool for studying gene function, yet are often a limiting step in gene function identification. In this study we have developed a F. oxysporum high-efficiency gene-disruption strategy based on split-marker homologous recombination cassettes with dual selection and electroporation transformation. The method was efficiently used to delete three RNA-dependent RNA polymerase (RdRP) genes. The gene-disruption cassettes of three genes can be constructed simultaneously within a short time using this technique. The optimal condition for electroporation is 10μF capacitance, 300Ω resistance, 4kV/cm field strength, with 1μg of DNA (gene-disruption cassettes). Under these optimal conditions, we were able to obtain 95 transformants per μg DNA. And after positive-negative selection, the transformants were efficiently screened by PCR, screening efficiency averaged 85%: 90% (RdRP1), 85% (RdRP2) and 77% (RdRP3). This gene-disruption strategy should pave the way for high throughout genetic analysis in F. oxysporum. Copyright © 2014 Elsevier GmbH. All rights reserved.

Electroporating Fields Target Oxidatively Damaged Areas in the Cell Membrane

PubMed Central

Vernier, P. Thomas; Levine, Zachary A.; Wu, Yu-Hsuan; Joubert, Vanessa; Ziegler, Matthew J.; Mir, Lluis M.; Tieleman, D. Peter

2009-01-01

Reversible electropermeabilization (electroporation) is widely used to facilitate the introduction of genetic material and pharmaceutical agents into living cells. Although considerable knowledge has been gained from the study of real and simulated model membranes in electric fields, efforts to optimize electroporation protocols are limited by a lack of detailed understanding of the molecular basis for the electropermeabilization of the complex biomolecular assembly that forms the plasma membrane. We show here, with results from both molecular dynamics simulations and experiments with living cells, that the oxidation of membrane components enhances the susceptibility of the membrane to electropermeabilization. Manipulation of the level of oxidative stress in cell suspensions and in tissues may lead to more efficient permeabilization procedures in the laboratory and in clinical applications such as electrochemotherapy and electrotransfection-mediated gene therapy. PMID:19956595

Using a nanopore for single molecule detection and single cell transfection.

PubMed

Nelson, Edward M; Kurz, Volker; Shim, Jiwook; Timp, Winston; Timp, Gregory

2012-07-07

We assert that it is possible to trap and identify proteins, and even (conceivably) manipulate proteins secreted from a single cell (i.e. the secretome) through transfection via electroporation by exploiting the exquisite control over the electrostatic potential available in a nanopore. These capabilities may be leveraged for single cell analysis and transfection with single molecule resolution, ultimately enabling a careful scrutiny of tissue heterogeneity.

An experimental system for controlled exposure of biological samples to electrostatic discharges.

PubMed

Marjanovič, Igor; Kotnik, Tadej

2013-12-01

Electrostatic discharges occur naturally as lightning strokes, and artificially in light sources and in materials processing. When an electrostatic discharge interacts with living matter, the basic physical effects can be accompanied by biophysical and biochemical phenomena, including cell excitation, electroporation, and electrofusion. To study these phenomena, we developed an experimental system that provides easy sample insertion and removal, protection from airborne particles, observability during the experiment, accurate discharge origin positioning, discharge delivery into the sample either through an electric arc with adjustable air gap width or through direct contact, and reliable electrical insulation where required. We tested the system by assessing irreversible electroporation of Escherichia coli bacteria (15 mm discharge arc, 100 A peak current, 0.1 μs zero-to-peak time, 0.2 μs peak-to-halving time), and gene electrotransfer into CHO cells (7 mm discharge arc, 14 A peak current, 0.5 μs zero-to-peak time, 1.0 μs peak-to-halving time). Exposures to natural lightning stroke can also be studied with this system, as due to radial current dissipation, the conditions achieved by a stroke at a particular distance from its entry are also achieved by an artificial discharge with electric current downscaled in magnitude, but similar in time course, correspondingly closer to its entry. © 2013.

A method of combined single-cell electrophysiology and electroporation.

PubMed

Graham, Lyle J; Del Abajo, Ricardo; Gener, Thomas; Fernandez, Eduardo

2007-02-15

This paper describes a method of extracellular recording and subsequent electroporation with the same electrode in single retinal ganglion cells in vitro. We demonstrate anatomical identification of neurons whose receptive fields were measured quantitatively. We discuss how this simple method should also be applicable for the delivery of a variety of intracellular agents, including gene delivery, to physiologically characterized neurons, both in vitro and in vivo.

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

PubMed

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

2015-04-07

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 patch-clamp artifact. In short, the response of DC-3F cells to strong pulsed electric fields was separated into a transient electroporation and a persistent permeabilization. The latter dominates postpulse membrane properties but to date has not been addressed by electroporation theory or MD simulations. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The future of human DNA vaccines

PubMed Central

Li, Lei; Saade, Fadi; Petrovsky, Nikolai

2012-01-01

DNA vaccines have evolved greatly over the last 20 years since their invention, but have yet to become a competitive alternative to conventional protein or carbohydrate based human vaccines. Whilst safety concerns were an initial barrier, the Achilles heel of DNA vaccines remains their poor immunogenicity when compared to protein vaccines. A wide variety of strategies have been developed to optimize DNA vaccine immunogenicity, including codon optimization, genetic adjuvants, electroporation and sophisticated prime-boost regimens, with each of these methods having its advantages and limitations. Whilst each of these methods has contributed to incremental improvements in DNA vaccine efficacy, more is still needed if human DNA vaccines are to succeed commercially. This review foresees a final breakthrough in human DNA vaccines will come from application of the latest cutting-edge technologies, including “epigenetics” and “omics” approaches, alongside traditional techniques to improve immunogenicity such as adjuvants and electroporation, thereby overcoming the current limitations of DNA vaccines in humans PMID:22981627

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

PubMed

Kos, Bor; Voigt, Peter; Miklavcic, Damijan; Moche, Michael

2015-09-01

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. The manufacturer of the only commercially available pulse generator for IRE recommends a voltage-to-distance ratio of 1500 to 1700 V/cm for treating tumors in the liver. However, major blood vessels can influence the electric field distribution. We present a method for treatment planning of IRE which takes the influence of blood vessels on the electric field into account; this is illustrated on a treatment of 48-year-old patient with a metastasis near the remaining hepatic vein after a right side hemi-hepatectomy. Output of the numerical treatment planning method shows that a 19.9 cm3 irreversible electroporation lesion was generated and the whole tumor was covered with at least 900 V/cm. This compares well with the volume of the hypodense lesion seen in contrast enhanced CT images taken after the IRE treatment. A significant temperature raise occurs near the electrodes. However, the hepatic vein remains open after the treatment without evidence of tumor recurrence after 6 months. Treatment planning using accurate computer models was recognized as important for electrochemotherapy and irreversible electroporation. An important finding of this study was, that the surface of the electrodes heat up significantly. Therefore the clinical user should generally avoid placing the electrodes less than 4 mm away from risk structures when following recommendations of the manufacturer.

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

PubMed Central

Kos, Bor; Voigt, Peter; Miklavcic, Damijan; Moche, Michael

2015-01-01

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. Materials and methods. The manufacturer of the only commercially available pulse generator for IRE recommends a voltage-to-distance ratio of 1500 to 1700 V/cm for treating tumors in the liver. However, major blood vessels can influence the electric field distribution. We present a method for treatment planning of IRE which takes the influence of blood vessels on the electric field into account; this is illustrated on a treatment of 48-year-old patient with a metastasis near the remaining hepatic vein after a right side hemi-hepatectomy. Results Output of the numerical treatment planning method shows that a 19.9 cm3 irreversible electroporation lesion was generated and the whole tumor was covered with at least 900 V/cm. This compares well with the volume of the hypodense lesion seen in contrast enhanced CT images taken after the IRE treatment. A significant temperature raise occurs near the electrodes. However, the hepatic vein remains open after the treatment without evidence of tumor recurrence after 6 months. Conclusions Treatment planning using accurate computer models was recognized as important for electrochemotherapy and irreversible electroporation. An important finding of this study was, that the surface of the electrodes heat up significantly. Therefore the clinical user should generally avoid placing the electrodes less than 4 mm away from risk structures when following recommendations of the manufacturer. PMID:26401128

[Electroporation of sperm to introduce foreign DNA into the genome of Pinctada maxima (Jameson)].

PubMed

Hu, W; Yu, D H; Wang, Y P; Wu, K C; Zhu, Z Y

2000-03-01

Gene transfer was investigated in marine molluscs via electroporated sperm. Sperm of P. maxima (J.) was incubated with linear "all-fish" growth hormone gene (pCAgcGH and pCAgcGHc) for 30 min. Then, mature eggs were in-vitro fertilized with the sperm cells treated with electroporation at 10 kV and 2(7) pulses of six cycles. DNA was extracted from spat and analyzed by PCR and southern blot. The results indicated that the foreign DNA had been transferred into the genome of experimental molluscs. The transgenetic ration was 5.6%, 20% and 50% when 2 micrograms/mL, 6 micrograms/mL and 18 micrograms/mL of foreign DNA was used, respectively. It is suggested that the transferred efficiency is correlated with the amount of the foreign DNA.

Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells.

PubMed

Hornstein, Benjamin D; Roman, Dany; Arévalo-Soliz, Lirio M; Engevik, Melinda A; Zechiedrich, Lynn

2016-01-01

The ability to produce extremely small and circular supercoiled vectors has opened new territory for improving non-viral gene therapy vectors. In this work, we compared transfection of supercoiled DNA vectors ranging from 383 to 4,548 bp, each encoding shRNA against GFP under control of the H1 promoter. We assessed knockdown of GFP by electroporation into HeLa cells. All of our vectors entered cells in comparable numbers when electroporated with equal moles of DNA. Despite similar cell entry, we found length-dependent differences in how efficiently the vectors knocked down GFP. As vector length increased up to 1,869 bp, GFP knockdown efficiency per mole of transfected DNA increased. From 1,869 to 4,257 bp, GFP knockdown efficiency per mole was steady, then decreased with increasing vector length. In comparing GFP knockdown with equal masses of vectors, we found that the shorter vectors transfect more efficiently per nanogram of DNA transfected. Our results rule out cell entry and DNA mass as determining factors for gene knockdown efficiency via electroporation. The length-dependent effects we have uncovered are likely explained by differences in nuclear translocation or transcription. These data add an important step towards clinical applications of non-viral vector delivery.

Safety and tolerability of the Easy Vax™ clinical epidermal electroporation system in healthy adults.

PubMed

El-Kamary, Samer S; Billington, Melissa; Deitz, Stephen; Colby, Elaina; Rhinehart, Howard; Wu, Yukun; Blackwelder, William; Edelman, Robert; Lee, Albert; King, Alan

2012-01-01

DNA vaccines are cost-effective and versatile, though intracellular delivery has been challenging in humans. Alternative delivery modalities such as electroporation have demonstrated improved immune responses, but are painful. In this single-center, double-blind, medical device trial, we evaluated the safety and tolerability of Easy Vax™ dermal electroporation system, alone (without DNA) in healthy adults. Three randomized protocol doses were administered to 10 subjects (80% white, 60% female, mean age: 32.1 years) in each of two areas (total of six doses). Two subjects complained of shooting pain, burning and/or tingling when doses were administered to the forearm region, but not the lateral deltoid regions. Subsequent doses for the remaining eight subjects were restricted to the deltoid regions only. Tolerability pain scores never exceeded 3 of 10 in the 11-Point Pain Rating scale, and 12 of 100 in the Visual Analog Scale (VAS), and lower in follow-up evaluations (P < 0.0001), with no significant difference between the three dosing protocols. Electrical properties of the skin, measured automatically by the device, showed no correlation between pain intensity and skin conductance. In conclusion, the Easy Vax™ electroporation device is safe and well tolerated when administered over the lateral deltoid skin regions in healthy volunteers.

Transformation of Escherichia coli with large DNA molecules by electroporation.

PubMed Central

Sheng, Y; Mancino, V; Birren, B

1995-01-01

We have examined bacterial electroporation with a specific interest in the transformation of large DNA, i.e. molecules > 100 kb. We have used DNA from bacterial artificial chromosomes (BACs) ranging from 7 to 240 kb, as well as BAC ligation mixes containing a range o different sized molecules. The efficiency of electroporation with large DNA is strongly dependent on the strain of Escherichia coli used; strains which offer comparable efficiencies for 7 kb molecules differ in their uptake of 240 kb DNA by as much as 30-fold. Even with a host strain that transforms relatively well with large DNA, transformation efficiency drops dramatically with increasing size of the DNA. Molecules of 240 kb transform approximately 30-fold less well, on a molar basis, than molecules of 80 kb. Maximum transformation of large DNA occurs with different voltage gradients and with different time constants than are optimal for smaller DNA. This provides the opportunity to increase the yield of transformants which have taken up large DNA relative to the number incorporating smaller molecules. We have demonstrated that conditions may be selected which increase the average size of BAC clones generated by electroporation and compare the overall efficiency of each of the conditions tested. Images PMID:7596828

Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells

PubMed Central

Hornstein, Benjamin D.; Roman, Dany; Arévalo-Soliz, Lirio M.; Engevik, Melinda A.

2016-01-01

The ability to produce extremely small and circular supercoiled vectors has opened new territory for improving non-viral gene therapy vectors. In this work, we compared transfection of supercoiled DNA vectors ranging from 383 to 4,548 bp, each encoding shRNA against GFP under control of the H1 promoter. We assessed knockdown of GFP by electroporation into HeLa cells. All of our vectors entered cells in comparable numbers when electroporated with equal moles of DNA. Despite similar cell entry, we found length-dependent differences in how efficiently the vectors knocked down GFP. As vector length increased up to 1,869 bp, GFP knockdown efficiency per mole of transfected DNA increased. From 1,869 to 4,257 bp, GFP knockdown efficiency per mole was steady, then decreased with increasing vector length. In comparing GFP knockdown with equal masses of vectors, we found that the shorter vectors transfect more efficiently per nanogram of DNA transfected. Our results rule out cell entry and DNA mass as determining factors for gene knockdown efficiency via electroporation. The length-dependent effects we have uncovered are likely explained by differences in nuclear translocation or transcription. These data add an important step towards clinical applications of non-viral vector delivery. PMID:27918590

Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution.

PubMed

Yamashiro, Sawako; Watanabe, Naoki

2017-07-06

Live-cell single-molecule imaging was introduced more than a decade ago, and has provided critical information on remodeling of the actin cytoskeleton, the motion of plasma membrane proteins, and dynamics of molecular motor proteins. Actin remodeling has been the best target for this approach because actin and its associated proteins stop diffusing when assembled, allowing visualization of single-molecules of fluorescently-labeled proteins in a state specific manner. The approach based on this simple principle is called Single-Molecule Speckle (SiMS) microscopy. For instance, spatiotemporal regulation of actin polymerization and lifetime distribution of actin filaments can be monitored directly by tracking actin SiMS. In combination with fluorescently labeled probes of various actin regulators, SiMS microscopy has contributed to clarifying the processes underlying recycling, motion and remodeling of the live-cell actin network. Recently, we introduced an electroporation-based method called eSiMS microscopy, with high efficiency, easiness and improved spatiotemporal precision. In this review, we describe the application of live-cell single-molecule imaging to cellular actin dynamics and discuss the advantages of eSiMS microscopy over previous SiMS microscopy.

Micro-magnetic Structures for Biological Applications

NASA Astrophysics Data System (ADS)

Howdyshell, Marci L.

Developments in single-molecule and single-cell experiments over the past century have provided researchers with many tools to probe the responses of cells to stresses such as physical force or to the injection of foreign genes. Often these techniques target the cell membrane, although many are now advancing to probe within the cell. As these techniques are improved upon and the investigations advance toward clinical applications, it has become more critical to achieve high-throughput outcomes which in turn lead to statistically significant results. The technologies developed in this thesis are targeted at transfecting large populations of cells with controlled doses of specific exogenic material without adversely affecting cell viability. Underlying this effort is a platform of lithographically patterned ferromagnetic thin films capable of remotely manipulating and localizing magnetic microbeads attached to biological entities. A novel feature of this approach, as demonstrated here with both DNA and cells, is the opportunity for multiplexed operations on targeted biological specimens. This thesis includes two main thrusts: (1) the advancement of the trapping platforms through experimental verification of mathematical models providing the energy landscapes associated with the traps and (2) implementation of the platform as a basis for rapid and effective high-throughput microchannel and nanochannel cell electroporation devices. The electroporation devices have, in our studies, not only been demonstrated to sustain cell viability with extremely low cell mortality rates, but are also found to be effective for various types of cells. The advances over current electroporation technologies that are achieved in these efforts demonstrate the potential for detection of mRNA expression in heterogeneous cell populations and probing intracellular responses to the introduction of foreign genes into cells.

Temporal Control of Gene Expression by Combining Electroporation and the Tetracycline Inducible Systems in Vertebrate Embryos

NASA Astrophysics Data System (ADS)

Dubrulle, Julien; Pourquié, Olivier

The electroporation technique has revolutionized vertebrate embryology. It has greatly contributed to our understanding of how genes and proteins can interact and regulate various aspects of vertebrate development in the last decade. This technique provides an efficient way to transfect embryonic cells in vivo with exogenous DNA by cre ating transient holes in the plasma membrane with short, squared electric pulses of low voltage (Itasaki et al., 1999; Momose et al., 1999; Muramatsu et al., 1997; Nakamura et al., 2004; Ogura, 2002). It has been particularly well-developed in the chick model since the large size of the embryo and its easy accessibility enables to target specific tissues with great precision. With the electroporation, it is possible to precisely choose which type of cells to transfect by performing a local injection of DNA close to the cells of interest, followed by the application of a small current through the targeted area. To date, all three germ layers — endoderm, mesoderm and ectoderm — as well as an increasing number of differentiated structures have been efficiently transfected (Dubrulle et al., 2001; Grapin-Botton et al., 2001; Itasaki et al., 1999; Luo and Redies, 2005; Scaal et al., 2004) and the continuous improvement in electrode design makes it even possible to aim at sub-populations of cells within a given tissue. In addition to this spatial precision, the technique also allows great temporal precision; any stage of development, ranging from pre-gastrulation stage to adulthood can be reached as long as the cells or structures are accessible for local DNA injection and electrode placement (Bigey et al., 2002; Iimura and Pourquie, 2006).

Apparatus and method for transforming living cells

DOEpatents

Okandan, Murat; Galambos, Paul C.

2003-11-11

An apparatus and method are disclosed for in vitro transformation of living cells. The apparatus, which is formed as a microelectromechanical device by surface micromachining, can be used to temporarily disrupt the cell walls or membrane of host cells one at a time so that a particular substance (e.g. a molecular tag, nucleic acid, bacteria, virus etc.) can be introduced into the cell. Disruption of the integrity of the host cells (i.e. poration) can be performed mechanically or electrically, or by both while the host cells are contained within a flow channel. Mechanical poration is possible using a moveable member which has a pointed or serrated edge and which is driven by an electrostatic actuator to abrade, impact or penetrate the host cell. Electroporation is produced by generating a relatively high electric field across the host cell when the host cell is located in the flow channel between a pair of electrodes having a voltage applied therebetween.

Irreversible Electroporation for Prostate Cancer as Salvage Treatment Following Prior Radiation and Cryotherapy.

PubMed

Murray, Katie S; Akin, Oguz; Coleman, Jonathan A

2017-01-01

Salvage treatment options after localized primary treatment failure of prostate cancer are limited and associated with risk for serious complications. We report on the management details of a 57-year-old African American man treated with partial-gland ablation using irreversible electroporation following local recurrence after brachytherapy and prior salvage cryoablation. Therapeutic and functional outcomes were assessed by conventional means, including serum prostate-specific antigen values and prostate biopsy results.

Highly Efficient Electroporation-mediated Transformation into Edible Mushroom Flammulina velutipes

PubMed Central

Kim, Jong Kun; Park, Young Jin; Kong, Won Sik

2010-01-01

In this study, we developed an efficient electroporation-mediated transformation system featuring Flammulina velutipes. The flammutoxin (ftx) gene of F. velutipes was isolated by reverse transcription-PCR. pFTXHg plasmid was constructed using the partial ftx gene (410 bp) along with the hygromycin B phosphotransferase gene (hygB) downstream of the glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter. The plasmid was transformed into protoplasts of monokaryotic strain 4019-20 of F. velutipes by electroporation. High transformation efficiency was obtained with an electric-pulse of 1.25 kV/cm by using 177 transformants/µg of DNA in 1 × 107 protoplasts. PCR and Southern blot hybridization indicated that a single copy of the plasmid DNA was inserted at different locations in the F. velutipes genome by non-homologous recombination. Therefore, this transformation system could be used as a useful tool for gene function analysis of F. velutipes. PMID:23956676

Highly Efficient Electroporation-mediated Transformation into Edible Mushroom Flammulina velutipes.

PubMed

Kim, Jong Kun; Park, Young Jin; Kong, Won Sik; Kang, Hee Wan

2010-12-01

In this study, we developed an efficient electroporation-mediated transformation system featuring Flammulina velutipes. The flammutoxin (ftx) gene of F. velutipes was isolated by reverse transcription-PCR. pFTXHg plasmid was constructed using the partial ftx gene (410 bp) along with the hygromycin B phosphotransferase gene (hygB) downstream of the glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter. The plasmid was transformed into protoplasts of monokaryotic strain 4019-20 of F. velutipes by electroporation. High transformation efficiency was obtained with an electric-pulse of 1.25 kV/cm by using 177 transformants/µg of DNA in 1 × 10(7) protoplasts. PCR and Southern blot hybridization indicated that a single copy of the plasmid DNA was inserted at different locations in the F. velutipes genome by non-homologous recombination. Therefore, this transformation system could be used as a useful tool for gene function analysis of F. velutipes.

Kinetics of ultraweak light emission from human erythroleukemia K562 cells upon electroporation.

PubMed

Maccarrone, M; Fantini, C; Agrò, A F; Rosato, N

1998-11-11

Electroporation involves the application of an electric pulse that creates transient aqueous channels (electropores) across the lipid bilayer membranes. Here, we describe an instrument set up suitable to record ultraweak light emission from human erythroleukemia K562 cells during and immediately after delivery of electric pulses. Most of light was emitted in the first seconds after each pulse, following a complex decay which can be fitted by a double exponential equation characterized by two different time constants (T1 and T2), both in the order of seconds. T1 was approximately 10-fold shorter than T2 and both time constants were dependent on field strength of the electric pulse. The effect of various antioxidants on the amount of emitted photons and on T1 and T2 values was investigated, in order to shed some light on the chemical species responsible for cellular luminescence.

Microscale Symmetrical Electroporator Array as a Versatile Molecular Delivery System

NASA Astrophysics Data System (ADS)

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.

Toward establishing model organisms for marine protists: Successful transfection protocols for Parabodo caudatus (Kinetoplastida: Excavata).

PubMed

Gomaa, Fatma; Garcia, Paulo A; Delaney, Jennifer; Girguis, Peter R; Buie, Cullen R; Edgcomb, Virginia P

2017-09-01

We developed protocols for, and demonstrated successful transfection of, the free-living kinetoplastid flagellate Parabodo caudatus with three plasmids carrying a fluorescence reporter gene (pEF-GFP with the EF1 alpha promoter, pUB-GFP with Ubiquitin C promoter, and pEYFP-Mitotrap with CMV promoter). We evaluated three electroporation approaches: (1) a square-wave electroporator designed for eukaryotes, (2) a novel microfluidic transfection system employing hydrodynamically-controlled electric field waveforms, and (3) a traditional exponential decay electroporator. We found the microfluidic device provides a simple and efficient platform to quickly test a wide range of electric field parameters to find the optimal set of conditions for electroporation of target species. It also allows for processing large sample volumes (>10 ml) within minutes, increasing throughput 100 times over cuvettes. Fluorescence signal from the reporter gene was detected a few hours after transfection and persisted for 3 days in cells transfected by pEF-GFP and pUB-GFP plasmids and for at least 5 days post-transfection for cells transfected with pEYFP-Mitotrap. Expression of the reporter genes (GFP and YFP) was also confirmed using reverse transcription-PCR (RT-PCR). This work opens the door for further efforts with this taxon and close relatives toward establishing model systems for genome editing. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

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

PubMed

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.

Nanosecond electric pulses modulate skeletal muscle calcium dynamics and contraction

NASA Astrophysics Data System (ADS)

Valdez, Chris; Jirjis, Michael B.; Roth, Caleb C.; Barnes, Ronald A.; Ibey, Bennett L.

2017-02-01

Irreversible electroporation therapy is utilized to remove cancerous tissues thru the delivery of rapid (250Hz) and high voltage (V) (1,500V/cm) electric pulses across microsecond durations. Clinical research demonstrated that bipolar (BP) high voltage microsecond pulses opposed to monophasic waveforms relieve muscle contraction during electroporation treatment. Our group along with others discovered that nanosecond electric pulses (nsEP) can activate second messenger cascades, induce cytoskeletal rearrangement, and depending on the nsEP duration and frequency, initiate apoptotic pathways. Of high interest across in vivo and in vitro applications, is how nsEP affects muscle physiology, and if nuances exist in comparison to longer duration electroporation applications. To this end, we exposed mature skeletal muscle cells to monopolar (MP) and BP nsEP stimulation across a wide range of electric field amplitudes (1-20 kV/cm). From live confocal microscopy, we simultaneously monitored intracellular calcium dynamics along with nsEP-induced muscle movement on a single cell level. In addition, we also evaluated membrane permeability with Yo-PRO-1 and Propidium Iodide (PI) across various nsEP parameters. The results from our findings suggest that skeletal muscle calcium dynamics, and nsEP-induced contraction exhibit exclusive responses to both MP and BP nsEP exposure. Overall the results suggest in vivo nsEP application may elicit unique physiology and field applications compared to longer pulse duration electroporation.

Investigating membrane nanoporation induced by bipolar pulsed electric fields via second harmonic generation

NASA Astrophysics Data System (ADS)

Moen, E. K.; Ibey, B. L.; Beier, H. T.; Armani, A. M.

2016-09-01

Electric pulses have become an effective tool for transporting cargo (DNA, drugs, etc.) across cell membranes. This enhanced transport is believed to occur through temporary pores formed in the plasma membrane. Traditionally, millisecond duration, monopolar (MP) pulses are used for electroporation, but bipolar (BP) pulses have proven equally effective as MP pulses with the added advantage of less cytotoxicity. With the goal of further reducing cytotoxic effects and inducing non-thermal, intra-cellular effects, researchers began investigating reduced pulse durations, pushing into the nanosecond regime. Cells exposed to these MP, nanosecond pulsed electric fields (nsPEFs) have shown increased repairable membrane permeability and selective channel activation. However, attempts to improve this further by moving to the BP pulse regime has proven unsuccessful. In the present work, we use second harmonic generation imaging to explore the structural effects of bipolar nsPEFs on the plasma membrane. By varying the temporal spacing between the pulse phases over several orders of magnitude and comparing the response to a single MP case, we systematically examine the disparity in cellular response. Our circuit-based model predicts that, as the temporal spacing increases several orders of magnitude, nanoporation increases and eventually exceeds the MP case. On the whole, our experimental data agree with this assertion; however, a detailed analysis of the data sets demonstrates that biological processes may play a larger role in the observed response than previously thought, dominating the effect for temporal spacing up to 5 μs. These findings could ultimately lead to understanding the biophysical mechanism underlying all electroporation.

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

NASA Astrophysics Data System (ADS)

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-economic profile of EP compares favourably with other drug and DNA delivery methods.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons.

PubMed

Eid, Lara; Lachance, Mathieu; Hickson, Gilles; Rossignol, Elsa

2018-04-20

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Passive and electro-assisted delivery of hydrogel nanoparticles in solid tumors, visualized by optical and magnetic resonance imaging in vivo.

PubMed

Bakalova, Rumiana; Nikolova, Biliana; Murayama, Shuhei; Atanasova, Severina; Zhelev, Zhivko; Aoki, Ichio; Kato, Masaru; Tsoneva, Iana; Saga, Tsuneo

2016-01-01

The present study describes a development of nanohydrogel, loaded with QD(705) and manganese (QD(705)@Nanogel and QD(705)@Mn@Nanogel), and its passive and electro-assisted delivery in solid tumors, visualized by fluorescence imaging and magnetic resonance imaging (MRI) on colon cancer-grafted mice as a model. QD(705)@Nanogel was delivered passively predominantly into the tumor, which was visualized in vivo and ex vivo using fluorescent imaging. The fluorescence intensity increased gradually within 30 min after injection, reached a plateau between 30 min and 2 h, and decreased gradually to the baseline within 24 h. The fluorescence intensity in the tumor area was about 2.5 times higher than the background fluorescence. A very weak fluorescent signal was detected in the liver area, but not in the areas of the kidneys or bladder. This result was in contrast with our previous study, indicating that FITC@Mn@Nanogel did not enter into the tumor and was detected rapidly in the kidney and bladder after i.v. injection [J. Mater. Chem. B 2013, 1, 4932-4938]. We found that the embedding of a hard material (as QD) in nanohydrogel changes the physical properties of the soft material (decreases the size and negative charge and changes the shape) and alters its pharmacodynamics. Electroporation facilitated the delivery of the nanohydrogel in the tumor tissue, visualized by fluorescent imaging and MRI. Strong signal intensity was recorded in the tumor area shortly after the combined treatment (QD@Mn@Nanogel + electroporation), and it was observed even 48 h after the electroporation. The data demonstrate more effective penetration of the nanoparticles in the tumor due to the increased permeability of blood vessels at the electroporated area. There was no rupture of blood vessels after electroporation, and there were no artifacts in the images due to a bleeding.

The future of human DNA vaccines.

PubMed

Li, Lei; Saade, Fadi; Petrovsky, Nikolai

2012-12-31

DNA vaccines have evolved greatly over the last 20 years since their invention, but have yet to become a competitive alternative to conventional protein or carbohydrate based human vaccines. Whilst safety concerns were an initial barrier, the Achilles heel of DNA vaccines remains their poor immunogenicity when compared to protein vaccines. A wide variety of strategies have been developed to optimize DNA vaccine immunogenicity, including codon optimization, genetic adjuvants, electroporation and sophisticated prime-boost regimens, with each of these methods having its advantages and limitations. Whilst each of these methods has contributed to incremental improvements in DNA vaccine efficacy, more is still needed if human DNA vaccines are to succeed commercially. This review foresees a final breakthrough in human DNA vaccines will come from application of the latest cutting-edge technologies, including "epigenetics" and "omics" approaches, alongside traditional techniques to improve immunogenicity such as adjuvants and electroporation, thereby overcoming the current limitations of DNA vaccines in humans. Copyright © 2012 Elsevier B.V. All rights reserved.

Production of a full-length infectious GFP-tagged cDNA clone of Beet mild yellowing virus for the study of plant-polerovirus interactions.

PubMed

Stevens, Mark; Viganó, Felicita

2007-04-01

The full-length cDNA of Beet mild yellowing virus (Broom's Barn isolate) was sequenced and cloned into the vector pLitmus 29 (pBMYV-BBfl). The sequence of BMYV-BBfl (5721 bases) shared 96% and 98% nucleotide identity with the other complete sequences of BMYV (BMYV-2ITB, France and BMYV-IPP, Germany respectively). Full-length capped RNA transcripts of pBMYV-BBfl were synthesised and found to be biologically active in Arabidopsis thaliana protoplasts following electroporation or PEG inoculation when the protoplasts were subsequently analysed using serological and molecular methods. The BMYV sequence was modified by inserting DNA that encoded the jellyfish green fluorescent protein (GFP) into the P5 gene close to its 3' end. A. thaliana protoplasts electroporated with these RNA transcripts were biologically active and up to 2% of transfected protoplasts showed GFP-specific fluorescence. The exploitation of these cDNA clones for the study of the biology of beet poleroviruses is discussed.

Use of irreversible electroporation in unresectable pancreatic cancer

PubMed Central

2015-01-01

Irreversible electroporation is a non-thermal injury ablative modality that has been in clinical use since 2008 in the treatment of locally advanced soft tissue tumors. It has been reported to be utilized intraoperatively, laparoscopically or percutaneously. The method of action of IRE relies on a high voltage (maximum 3,000 volts) small microsecond pulse lengths (70 to 90 microseconds) to induce cell membrane porosity which leads to slow/protracted cell death over time. One of the largest unmet needs in oncology that IRE has been utilized is in locally advanced (stage III) pancreatic cancer. Recent studies have demonstrated the safety and palliation with encouraging improvement in overall survival. Its inherent limitation still remains tissue heterogeneity and the unique settings based on tumor histology and prior induction therapy. There remains a high technical demand of the end-user and the more extensive knowledge transfer which makes the learning curve longer in order to achieve appropriate and safe utilization. PMID:26151062

Electroporation of Mammalian Cells by Nanosecond Electric Field Oscillations and its Inhibition by the Electric Field Reversal

DTIC Science & Technology

2015-09-08

Report 3. DATES COVERED (From – To) March 2013 to July 2015 4 . TITLE AND SUBTITLE Electroporation of mammalian cells by nanosecond electric field...NEFO was a damped sine wave with 140 ns first phase duration at 50% height; the peak amplitude of phases 2- 4 decreased to 35%, 12%, and 7% of the...first phase. This waveform was rectified to produce unipolar NEFO by cutting off phases 2 and 4 . Membrane permeabilization was quantified in CHO and

Smallpox DNA Vaccine Delivered by Novel Skin Electroporation Device Protects Mice Against Intranasal Poxvirus Challenge

DTIC Science & Technology

2006-11-27

response being elicited by microneedle -mediated skin electroporation. 2006 Elsevier Ltd. All rights reserved. i o a p ( c o t t v H f r eywords...localized skin infection containing infectious virus (i.e., ock), the infection can spread to other sites on the body e.g., ocular autoinoculation) or to...plasmid DNA-coated microneedle arrays. Mice vaccinated with the 4pox DNA vaccine mounted robust antibody responses against the four immunogens-of-interest

Transient Expression of an LEDGF/p75 Chimera Retargets Lentivector Integration and Functionally Rescues in a Model for X-CGD

PubMed Central

Vets, Sofie; De Rijck, Jan; Brendel, Christian; Grez, Manuel; Bushman, Frederic; Debyser, Zeger; Gijsbers, Rik

2013-01-01

Retrovirus-based vectors are commonly used as delivery vehicles to correct genetic diseases because of their ability to integrate new sequences stably. However, adverse events in which vector integration activates proto-oncogenes, leading to clonal expansion and leukemogenesis hamper their application. The host cell-encoded lens epithelium-derived growth factor (LEDGF/p75) binds lentiviral integrase and targets integration to active transcription units. We demonstrated earlier that replacing the LEDGF/p75 chromatin interaction domain with an alternative DNA-binding protein could retarget integration. Here, we show that transient expression of the chimeric protein using mRNA electroporation efficiently redirects lentiviral vector (LV) integration in wild-type (WT) cells. We then employed this technology in a model for X-linked chronic granulomatous disease (X-CGD) using myelomonocytic PLB-985 gp91−/− cells. Following electroporation with mRNA encoding the LEDGF-chimera, the cells were treated with a therapeutic lentivector encoding gp91phox. Integration site analysis revealed retargeted integration away from genes and towards heterochromatin-binding protein 1β (CBX1)-binding sites, in regions enriched in marks associated with gene silencing. Nevertheless, gp91phox expression was stable for at least 6 months after electroporation and NADPH-oxidase activity was restored to normal levels as determined by superoxide production. Together, these data provide proof-of-principle that transient expression of engineered LEDGF-chimera can retarget lentivector integration and rescues the disease phenotype in a cell model, opening perspectives for safer gene therapy. PMID:23462964

Electrical Impedance Spectroscopy Study of Biological Tissues

PubMed Central

Dean, D.A.; Ramanathan, T.; Machado, D.; Sundararajan, R.

2008-01-01

The objective of this study was to investigate the electrical impedance properties of rat lung and other tissues ex vivo using Electrical Impedance Spectroscopy. Rat lungs (both electroporated and naïve (untreated)), and mesenteric vessels (naïve) were harvested from male Sprague-Dawley rats; their electrical impedance were measured using a Solartron 1290 impedance analyzer. Mouse lung and heart samples (naïve) were also studied. The resistance (Real Z, ohm) and the reactance (Im Z, negative ohm)) magnitudes and hence the Cole-Cole (Real Z versus Im Z) plots are different for the electroporated lung and the naive lung. The results confirm the close relationship between the structure and the functional characteristic. These also vary for the different biological tissues studied. The impedance values were higher at low frequencies compared to those at high frequencies. This study is of practical interest for biological applications of electrical pulses, such as electroporation, whose efficacy depends on cell type and its electrical impedance characteristics. PMID:19255614

Irreversible electroporation of locally advanced pancreatic neck/body adenocarcinoma

PubMed Central

2015-01-01

Objective Irreversible electroporation (IRE) of locally advanced pancreatic adenocarcinoma of the neck has been used to palliate appropriate stage 3 pancreatic cancers without evidence of metastasis and who have undergone appropriate induction therapy. Currently there has not been a standardized reported technique for pancreatic mid-body tumors for patient selection and intra-operative technique. Patients Subjects are patients with locally advanced pancreatic adenocarcinoma of the body/neck who have undergone appropriate induction chemotherapy for a reasonable duration. Main outcome measures Technique of open IRE of locally advanced pancreatic adenocarcinoma of the neck/body is described, with the emphasis on intra-operative ultrasound and intra-operative electroporation management. Results The technique of open IRE of the pancreatic neck/body with bracketing of the celiac axis and superior mesenteric artery with continuous intraoperative ultrasound imaging and consideration of intraoperative navigational system is described. Conclusions IRE of locally advanced pancreatic adenocarcinoma of the body/neck is feasible for appropriate patients with locally advanced unresectable pancreatic cancer. PMID:26029461

Characterization of carrier erythrocytes for biosensing applications

NASA Astrophysics Data System (ADS)

Bustamante López, Sandra C.; Meissner, Kenith E.

2017-09-01

Erythrocyte abundance, mobility, and carrying capacity make them attractive as a platform for blood analyte sensing as well as for drug delivery. Sensor-loaded erythrocytes, dubbed erythrosensors, could be reinfused into the bloodstream, excited noninvasively through the skin, and used to provide measurement of analyte levels in the bloodstream. Several techniques to load erythrocytes, thus creating carrier erythrocytes, exist. However, their cellular characteristics remain largely unstudied. Changes in cellular characteristics lead to removal from the bloodstream. We hypothesize that erythrosensors need to maintain native erythrocytes' (NEs) characteristics to serve as a long-term sensing platform. Here, we investigate two loading techniques and the properties of the resulting erythrosensors. For loading, hypotonic dilution requires a hypotonic solution while electroporation relies on electrical pulses to perforate the erythrocyte membrane. We analyze the resulting erythrosensor signal, size, morphology, and hemoglobin content. Although the resulting erythrosensors exhibit morphological changes, their size was comparable with NEs. The hypotonic dilution technique was found to load erythrosensors much more efficiently than electroporation, and the sensors were loaded throughout the volume of the erythrosensors. Finally, both techniques resulted in significant loss of hemoglobin. This study points to the need for continued development of loading techniques that better preserve NE characteristics.

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation.

PubMed

Montoya, Misty M; Ansel, K Mark

2017-04-13

CD4 + T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from naïve CD4 + T cells in vitro by activating them in the presence of the polarizing cytokines IL-1β, IL-6, IL-23, and TGFβ. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, RORγt, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Site-targeted non-viral gene delivery by direct DNA injection into the pancreatic parenchyma and subsequent in vivo electroporation in mice.

PubMed

Sato, Masahiro; Inada, Emi; Saitoh, Issei; Ohtsuka, Masato; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi

2013-11-01

The pancreas is considered an important gene therapy target because the organ is the site of several high burden diseases, including diabetes mellitus, cystic fibrosis, and pancreatic cancer. We aimed to develop an efficient in vivo gene delivery system using non-viral DNA. Direct intra-parenchymal injection of a solution containing circular plasmid pmaxGFP DNA was performed on adult anesthetized ICR female mice. The injection site was sandwiched with a pair of tweezer-type electrode disks, and electroporated using a square-pulse generator. Green fluorescent protein (GFP) expression within the injected pancreatic portion was observed one day after gene delivery. GFP expression reduced to baseline within a week of transfection. Application of voltages over 40 V resulted in tissue damage during electroporation. We demonstrate that electroporation is effective for safe and efficient transfection of pancreatic cells. This novel gene delivery method to the pancreatic parenchyma may find application in gene therapy strategies for pancreatic diseases and in investigation of specific gene function in situ. © 2013 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptions are made.

Lab-on-a-chip technologies for proteomic analysis from isolated cells.

PubMed

Sedgwick, H; Caron, F; Monaghan, P B; Kolch, W; Cooper, J M

2008-10-06

Lab-on-a-chip systems offer a versatile environment in which low numbers of cells and molecules can be manipulated, captured, detected and analysed. We describe here a microfluidic device that allows the isolation, electroporation and lysis of single cells. A431 human epithelial carcinoma cells, expressing a green fluorescent protein-labelled actin, were trapped by dielectrophoresis within an integrated lab-on-a-chip device containing saw-tooth microelectrodes. Using these same trapping electrodes, on-chip electroporation was performed, resulting in cell lysis. Protein release was monitored by confocal fluorescence microscopy.

Immunogenicity and malaria transmission reducing potency of Pfs48/45 and Pfs25 encoded by DNA vaccines administered by intramuscular electroporation.

PubMed

Datta, Dibyadyuti; Bansal, Geetha P; Gerloff, Dietlind L; Ellefsen, Barry; Hannaman, Drew; Kumar, Nirbhay

2017-01-05

Pfs48/45 and Pfs25 are leading candidates for the development of Plasmodium falciparum transmission blocking vaccines (TBV). Expression of Pfs48/45 in the erythrocytic sexual stages and presentation to the immune system during infection in the human host also makes it ideal for natural boosting. However, it has been challenging to produce a fully folded, functionally active Pfs48/45, using various protein expression platforms. In this study, we demonstrate that full-length Pfs48/45 encoded by DNA plasmids is able to induce significant transmission reducing immune responses. DNA plasmids encoding Pfs48/45 based on native (WT), codon optimized (SYN), or codon optimized and mutated (MUT1 and MUT2), to prevent any asparagine (N)-linked glycosylation were compared with or without intramuscular electroporation (EP). EP significantly enhanced antibody titers and transmission blocking activity elicited by immunization with SYN Pfs48/45 DNA vaccine. Mosquito membrane feeding assays also revealed improved functional immunogenicity of SYN Pfs48/45 (N-glycosylation sites intact) as compared to MUT1 or MUT2 Pfs48/45 DNA plasmids (all N-glycosylation sites mutated). Boosting with recombinant Pfs48/45 protein after immunization with each of the different DNA vaccines resulted in significant boosting of antibody response and improved transmission reducing capabilities of all four DNA vaccines. Finally, immunization with a combination of DNA plasmids (SYN Pfs48/45 and SYN Pfs25) also provides support for the possibility of combining antigens targeting different life cycle stages in the parasite during transmission through mosquitoes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Site-Directed Mutagenesis, in Vivo Electroporation and Mass Spectrometry in Search for Determinants of the Subcellular Targeting of Rab7b Paralogue in the Model Eukaryote Paramecium Octaurelia

PubMed Central

Wyroba, E.; Kwaśniak, P.; Miller, K.; Kobyłecki, K.; Osińska, M.

2016-01-01

Protein products of paralogous genes resulting from whole genome duplication may acquire new functions. The role of post-translational modifications (PTM) in proper targeting of Paramecium Rab7b paralogue (distinct from that of Rab7a directly involved in phagocytosis) was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala diminished the incorporation of [P32] by 37% and of [C14-]UDP-glucose by 24% into recombinant Rab7b_200 in comparison to the non-mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells in contrast to non-mutagenized recombinant Rab7b correctly incorporated in the cytostome area. Using nano LC-MS/MS to compare the peptide map of Rab7b with that after deglycosylation with a mixture of five enzymes of different specificity we identified a peptide ion at m/z=677.63+ representing a glycan group attached to Thr200. Based on its mass and quantitative assays with [P32] and [C14]UDP-glucose, the suggested composition of the adduct attached to Thr200 is (Hex)1(HexNAc)1(Phos)3 or (HexNAc)1 (Deoxyhexose)1 (Phos)1 (HexA)1. These data indicate that PTM of Thr200 located in the hypervariable C-region of Paramecium octaurelia Rab7b is crucial for the proper localization/function of this protein. Moreover, the two Rab7 paralogues differ also in another PTM: substantially more phosphorylated amino acid residues are in Rab7b than in Rab7a. PMID:27349314

Transient Expression of an LEDGF/p75 Chimera Retargets Lentivector Integration and Functionally Rescues in a Model for X-CGD.

PubMed

Vets, Sofie; De Rijck, Jan; Brendel, Christian; Grez, Manuel; Bushman, Frederic; Debyser, Zeger; Gijsbers, Rik

2013-03-05

Retrovirus-based vectors are commonly used as delivery vehicles to correct genetic diseases because of their ability to integrate new sequences stably. However, adverse events in which vector integration activates proto-oncogenes, leading to clonal expansion and leukemogenesis hamper their application. The host cell-encoded lens epithelium-derived growth factor (LEDGF/p75) binds lentiviral integrase and targets integration to active transcription units. We demonstrated earlier that replacing the LEDGF/p75 chromatin interaction domain with an alternative DNA-binding protein could retarget integration. Here, we show that transient expression of the chimeric protein using mRNA electroporation efficiently redirects lentiviral vector (LV) integration in wild-type (WT) cells. We then employed this technology in a model for X-linked chronic granulomatous disease (X-CGD) using myelomonocytic PLB-985 gp91(-/-) cells. Following electroporation with mRNA encoding the LEDGF-chimera, the cells were treated with a therapeutic lentivector encoding gp91(phox). Integration site analysis revealed retargeted integration away from genes and towards heterochromatin-binding protein 1β (CBX1)-binding sites, in regions enriched in marks associated with gene silencing. Nevertheless, gp91(phox) expression was stable for at least 6 months after electroporation and NADPH-oxidase activity was restored to normal levels as determined by superoxide production. Together, these data provide proof-of-principle that transient expression of engineered LEDGF-chimera can retarget lentivector integration and rescues the disease phenotype in a cell model, opening perspectives for safer gene therapy.Molecular Therapy - Nucleic Acids (2013) 2, e77; doi:10.1038/mtna.2013.4; published online 5 March 2013.

Physical non-viral gene delivery methods for tissue engineering.

PubMed

Mellott, Adam J; Forrest, M Laird; Detamore, Michael S

2013-03-01

The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that "fits-all" cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications.

Physical non-viral gene delivery methods for tissue engineering

PubMed Central

Mellott, Adam J.; Forrest, M. Laird; Detamore, Michael S.

2016-01-01

The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that “fits-all” cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications. PMID:23099792

In vivo electroporation enhances vaccine-mediated therapeutic control of human papilloma virus-associated tumors by the activation of multifunctional and effector memory CD8+ T cells.

PubMed

Sales, Natiely S; Silva, Jamile R; Aps, Luana R M M; Silva, Mariângela O; Porchia, Bruna F M M; Ferreira, Luís Carlos S; Diniz, Mariana O

2017-12-19

In vivo electroporation (EP) has reignited the clinical interest on DNA vaccines as immunotherapeutic approaches to control different types of cancer. EP has been associated with increased immune response potency, but its capacity in influencing immunomodulation remains unclear. Here we evaluated the impact of in vivo EP on the induction of cellular immune responses and therapeutic effects of a DNA vaccine targeting human papillomavirus-induced tumors. Our results demonstrate that association of EP with the conventional intramuscular administration route promoted a more efficient activation of multifunctional and effector memory CD8 + T cells with enhanced cytotoxic activity. Furthermore, EP increased tumor infiltration of CD8 + T cells and avoided tumor recurrences. Finally, our results demonstrated that EP promotes local migration of antigen presenting cells that enhances with vaccine co-delivery. Altogether the present evidences shed further light on the in vivo electroporation action and its impact on the immunogenicity of DNA vaccines. Copyright © 2017 Elsevier Ltd. All rights reserved.

Requirement of alveolar bone formation for eruption of rat molars

PubMed Central

Wise, Gary E.; He, Hongzhi; Gutierrez, Dina L.; Ring, Sherry; Yao, Shaomian

2011-01-01

Tooth eruption is a localized event that requires a dental follicle (DF) to regulate the resorption of alveolar bone to form an eruption pathway. During the intra-osseous phase of eruption, the tooth moves through this pathway. The mechanism or motive force that propels the tooth through this pathway is controversial but many studies have shown that alveolar bone growth at the base of the crypt occurs during eruption. To determine if this bone growth (osteogenesis) was causal, experiments were designed in which the expression of an osteogenic gene in the DF, bone morphogenetic protein-6 (BMP6), was inhibited by injection of the 1st mandibular molar of the rat with an siRNA targeted against BMP6. The injection was followed by electroporation to promote uptake of the siRNA. In 45 first molars injected, eruption either was delayed or completely inhibited (7 molars). In the impacted molars, an eruption pathway formed but bone growth at the base of the crypt was greatly reduced as compared to the erupted first molar controls. These studies show that alveolar bone growth at the base of the crypt is required for tooth eruption and that BMP6 may be an essential gene for promoting this growth. PMID:21896048

Comparison of cytotoxic T lymphocyte responses against pancreatic cancer induced by dendritic cells transfected with total tumor RNA and fusion hybrided with tumor cell

PubMed Central

Chen, Jiang; Li, Hong-Yu; Wang, Di; Shao, Xiao-Dong

2015-01-01

Pancreatic cancer (PC) is a deadly human malignancy. Dendritic cell (DC)-based immunotherapy with whole tumor antigens demonstrates potential efficiency in cancer treatment. Tumor RNA and tumor fusion hybrid cells are sources of whole tumor antigens for preparing DC tumor vaccines. However, the efficacy of these sources in eliciting immune responses against PC has not yet to be directly compared. In the present study, patient-derived PC cells and DCs were fused (DC–tumor hybrids) and primary cultured PC cell-derived total RNA was electroporated into autologous DCs (DC–tumor RNA). The antitumor immune responses induced by DC–tumor hybrids and DC–tumor RNA were compared directly. The results showed that both RNA and hybrid methodologies could induce tumor-specific cytotoxic T lymphocyte (CTL) responses, but pulsing DCs with total tumor RNA could induce a higher frequency of activated CTLs and T-helper cells than fusing DCs with autologous tumor cells. In addition, DC–tumor RNA triggered stronger autologous tumor cell lysis than DC–tumor hybrids. It could be concluded that DCs pulsed with whole tumor RNA are superior to those fused with tumor cells in priming anti-PC CTL responses. Electroporation with total tumor RNA may be more suitable for DC-based PC vaccination. PMID:25736302

Radiation Interaction with Therapeutic Drugs and Cell Membranes

NASA Astrophysics Data System (ADS)

Martin, Diana I.; Manaila, Elena N.; Moisescu, Mihaela I.; Savopol, Tudor D.; Kovacs, Eugenia A.; Cinca, Sabin A.; Matei, Constantin I.; Margaritescu, Irina D.; Iacob, Nicusor I.; Ighigeanu, Daniel I.; Craciun, Gabriela D.

2007-04-01

This transient permeabilized state of the cell membrane, named the ``cell electroporation'' (CE) can be used to increase cells uptake of drugs that do not readily pass cell membrane, thus enabling their cytotoxicity. The anticancer drugs, such as bleomycin (BL) and cisplatin, are the most candidates for the combined use with ionizing and non-ionizing radiation fields. The methods and installations for the cell electroporation by electron beam (EB) and microwave (MW) irradiation are presented. The viability tests of the human leukocytes under EB and MW exposure with/without the BL in the cell cultures are discussed.

Generating mammalian stable cell lines by electroporation.

PubMed

A Longo, Patti; Kavran, Jennifer M; Kim, Min-Sung; Leahy, Daniel J

2013-01-01

Expression of functional, recombinant mammalian proteins often requires expression in mammalian cells (see Single Cell Cloning of a Stable Mammalian Cell Line). If the expressed protein needs to be made frequently, it can be best to generate a stable cell line instead of performing repeated transient transfections into mammalian cells. Here, we describe a method to generate stable cell lines via electroporation followed by selection steps. This protocol will be limited to the CHO dhfr-Urlaub et al. (1983) and LEC1 cell lines, which in our experience perform the best with this method. Copyright © 2013 Elsevier Inc. All rights reserved.

Lab-on-a-chip technologies for proteomic analysis from isolated cells

PubMed Central

Sedgwick, H.; Caron, F.; Monaghan, P.B.; Kolch, W.; Cooper, J.M.

2008-01-01

Lab-on-a-chip systems offer a versatile environment in which low numbers of cells and molecules can be manipulated, captured, detected and analysed. We describe here a microfluidic device that allows the isolation, electroporation and lysis of single cells. A431 human epithelial carcinoma cells, expressing a green fluorescent protein-labelled actin, were trapped by dielectrophoresis within an integrated lab-on-a-chip device containing saw-tooth microelectrodes. Using these same trapping electrodes, on-chip electroporation was performed, resulting in cell lysis. Protein release was monitored by confocal fluorescence microscopy. PMID:18534931

pEGFP transfection into murine skeletal muscle by electrosonoporation

NASA Astrophysics Data System (ADS)

Tamošiūnas, Mindaugas; Jakovels, Dainis; Rubins, Uldis; Kadikis, Roberts; Petrovska, Ramona; Šatkauskas, Saulius

2017-12-01

In this study, we aimed to determine whether the combination of electroporation (EP) and ultrasound (US) waves (sonoporation) can affect the plasmid DNA transfection to mice tibialis cranialis muscle. Multispectral imaging technique combined with fluorescence spectroscopy point measurements has been used for the transcutaneous detection of enhanced green fluorescent protein (EGFP) fluorescence, providing information on location and duration of EGFP expression. We found that electrosonoporation, commonly enhancing pDNA transfection in vitro, had no positive effect on EGFP transfection efficiency increase in vivo with respect to electroporation alone. We presume that this may be associated with decreased viability of transfected fibers.

Ex vivo electroporation of retinal cells: a novel, high efficiency method for functional studies in primary retinal cultures

PubMed Central

Vergara, Maria Natalia; Gutierrez, Christian; O’Brien, David R.; Canto-Soler, Maria Valeria

2013-01-01

Primary retinal cultures constitute valuable tools not only for basic research on retinal cell development and physiology, but also for the identification of factors or drugs that promote cell survival and differentiation. In order to take full advantage of the benefits of this system it is imperative to develop efficient and reliable techniques for the manipulation of gene expression. However, achieving appropriate transfection efficiencies in these cultures has remained challenging. The purpose of this work was to develop and optimize a technique that would allow the transfection of chick retinal cells with high efficiency and reproducibility for multiple applications. We developed an ex vivo electroporation method applied to dissociated retinal cell cultures that offers a significant improvement over other currently available transfection techniques, increasing efficiency by five-fold. In this method, eyes were enucleated, devoid of RPE, and electroporated with GFP-encoding plasmids using custom-made electrodes. Electroporated retinas were then dissociated into single cells and plated in low density conditions, to be analyzed after 4 days of incubation. Parameters such as voltage and number of electric pulses, as well as plasmid concentration and developmental stage of the animal were optimized for efficiency. The characteristics of the cultures were assessed by morphology and immunocytochemistry, and cell viability was determined by ethidium homodimer staining. Cell imaging and counting was performed using an automated high-throughput system. This procedure resulted in transfection efficiencies in the order of 22–25 % of cultured cells, encompassing both photoreceptors and non-photoreceptor neurons, and without affecting normal cell survival and differentiation. Finally, the feasibility of the technique for cell-autonomous studies of gene function in a biologically relevant context was tested by carrying out gain and loss-of-function experiments for the transcription factor PAX6. Electroporation of a plasmid construct expressing PAX6 resulted in a marked upregulation in the expression levels of this protein that could be measured in the whole culture as well as cell-intrinsically. This was accompanied by a significant decrease in the percentage of cells differentiating as photoreceptors among the transfected population. Conversely, electroporation of an RNAi construct targeting PAX6 resulted in a significant decrease in the levels of this protein, with a concomitant increase in the proportion of photoreceptors. Taken together these results provide strong proof-of-principle of the suitability of this technique for genetic studies in retinal cultures. The combination of the high transfection efficiency obtained by this method with automated high-throughput cell analysis supplies the scientific community with a powerful system for performing functional studies in a cell-autonomous manner. PMID:23370269

Ex vivo electroporation of retinal cells: a novel, high efficiency method for functional studies in primary retinal cultures.

PubMed

Vergara, M Natalia; Gutierrez, Christian; O'Brien, David R; Canto-Soler, M Valeria

2013-04-01

Primary retinal cultures constitute valuable tools not only for basic research on retinal cell development and physiology, but also for the identification of factors or drugs that promote cell survival and differentiation. In order to take full advantage of the benefits of this system it is imperative to develop efficient and reliable techniques for the manipulation of gene expression. However, achieving appropriate transfection efficiencies in these cultures has remained challenging. The purpose of this work was to develop and optimize a technique that would allow the transfection of chick retinal cells with high efficiency and reproducibility for multiple applications. We developed an ex vivo electroporation method applied to dissociated retinal cell cultures that offers a significant improvement over other currently available transfection techniques, increasing efficiency by five-fold. In this method, eyes were enucleated, devoid of RPE, and electroporated with GFP-encoding plasmids using custom-made electrodes. Electroporated retinas were then dissociated into single cells and plated in low density conditions, to be analyzed after 4 days of incubation. Parameters such as voltage and number of electric pulses, as well as plasmid concentration and developmental stage of the animal were optimized for efficiency. The characteristics of the cultures were assessed by morphology and immunocytochemistry, and cell viability was determined by ethidium homodimer staining. Cell imaging and counting was performed using an automated high-throughput system. This procedure resulted in transfection efficiencies in the order of 22-25% of cultured cells, encompassing both photoreceptors and non-photoreceptor neurons, and without affecting normal cell survival and differentiation. Finally, the feasibility of the technique for cell-autonomous studies of gene function in a biologically relevant context was tested by carrying out gain and loss-of-function experiments for the transcription factor PAX6. Electroporation of a plasmid construct expressing PAX6 resulted in a marked upregulation in the expression levels of this protein that could be measured in the whole culture as well as cell-intrinsically. This was accompanied by a significant decrease in the percentage of cells differentiating as photoreceptors among the transfected population. Conversely, electroporation of an RNAi construct targeting PAX6 resulted in a significant decrease in the levels of this protein, with a concomitant increase in the proportion of photoreceptors. Taken together these results provide strong proof-of-principle of the suitability of this technique for genetic studies in retinal cultures. The combination of the high transfection efficiency obtained by this method with automated high-throughput cell analysis supplies the scientific community with a powerful system for performing functional studies in a cell-autonomous manner. Copyright © 2013 Elsevier Ltd. All rights reserved.

FGF and BMP derived from dorsal root ganglia regulate blastema induction in limb regeneration in Ambystoma mexicanum.

PubMed

Satoh, Akira; Makanae, Aki; Nishimoto, Yurie; Mitogawa, Kazumasa

2016-09-01

Urodele amphibians have a remarkable organ regeneration ability that is regulated by neural inputs. The identification of these neural inputs has been a challenge. Recently, Fibroblast growth factor (Fgf) and Bone morphogenic protein (Bmp) were shown to substitute for nerve functions in limb and tail regeneration in urodele amphibians. However, direct evidence of Fgf and Bmp being secreted from nerve endings and regulating regeneration has not yet been shown. Thus, it remained uncertain whether they were the nerve factors responsible for successful limb regeneration. To gather experimental evidence, the technical difficulties involved in the usage of axolotls had to be overcome. We achieved this by modifying the electroporation method. When Fgf8-AcGFP or Bmp7-AcGFP was electroporated into the axolotl dorsal root ganglia (DRG), GFP signals were detectable in the regenerating limb region. This suggested that Fgf8 and Bmp7 synthesized in neural cells in the DRG were delivered to the limbs through the long axons. Further knockdown experiments with double-stranded RNA interference resulted in impaired limb regeneration ability. These results strongly suggest that Fgf and Bmp are the major neural inputs that control the organ regeneration ability. Copyright © 2016 Elsevier Inc. All rights reserved.

DERMAL DRUG LEVELS OF ANTIBIOTIC (CEPHALEXIN) DETERMINED BY ELECTROPORATION AND TRANSCUTANEOUS SAMPLING (ETS) TECHNIQUE

PubMed Central

Sammeta, SM; Vaka, SRK; Murthy, S. Narasimha

2009-01-01

The purpose of this project was to assess the validity of a novel “Electroporation and transcutaneous sampling (ETS)” technique for sampling cephalexin from the dermal extracellular fluid (ECF). This work also investigated the plausibility of using cephalexin levels in the dermal ECF as a surrogate for the drug level in the synovial fluid. In vitro and in vivo studies were carried out using hair less rats to assess the workability of ETS. Cephalexin (20mg/kg) was administered i.v. through tail vein and the time course of drug concentration in the plasma was determined. In the same rats, cephalexin concentration in the dermal ECF was determined by ETS and microdialysis techniques. In a separate set of rats, only intraarticular microdialysis was carried out determine the time course of cephalexin concentration in synovial fluid. The drug concentration in the dermal ECF determined by ETS and microdialysis did not differ significantly from each other and so as were the pharmacokinetic parameters. The results provide validity to the ETS technique. Further, there was a good correlation (~0.9) between synovial fluid and dermal ECF levels of cephalexin indicating that dermal ECF levels could be used as a potential surrogate for cephalexin concentration in the synovial fluid. PMID:19067398

Selection of a novel peptide aptamer with high affinity for TiO2-nanoparticle through a direct electroporation with TiO2-binding phage complexes.

PubMed

Inoue, Ippei; Ishikawa, Yasuaki; Uraoka, Yukiharu; Yamashita, Ichiro; Yasueda, Hisashi

2016-11-01

We have developed an easy and rapid screening method of peptide aptamers with high affinity for a target material TiO 2 using M13 phage-display and panning procedure. In a selection step, the phage-substrate complexes and Escherichia coli cells were directly applied by electric pulse for electroporation, without separating the objective phages from the TiO 2 nanoparticles. Using this simple and rapid method, we obtained a novel peptide aptamer (named ST-1 with the sequence AYPQKFNNNFMS) with highly strong binding activity for TiO 2 . A cage-shaped protein fused with both ST-1 and an available carbon nanotube-affinity peptide was designed and produced in E. coli. The multi-functional supraprotein could efficiently mineralize a titanium-compound around the surface of single-wall carbon nanotubes (SWNTs), indicating that the ST-1 is valuable in the fabrication of nano-composite materials with titanium-compounds. The structural analysis of ST-1 variants indicated the importance of the N-terminal region (as a motif of AXPQKX 6 S) of the aptamer in the TiO 2 -binding activity. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Irreversible electroporation: Just another form of thermal therapy?

PubMed Central

van Gemert, Martin J C; Wagstaff, Peter G K; de Bruin, Daniel M; van Leeuwen, Ton G; van der Wal, Allard C; Heger, Michal; van der Geld, Cees W M

2015-01-01

Background Irreversible electroporation (IRE) is (virtually) always called non-thermal despite many reports showing that significant Joule heating occurs. Our first aim is to validate with mathematical simulations that IRE as currently practiced has a non-negligible thermal response. Our second aim is to present a method that allows simple temperature estimation to aid IRE treatment planning. Methods We derived an approximate analytical solution of the bio-heat equation for multiple 2-needle IRE pulses in an electrically conducting medium, with and without a blood vessel, and incorporated published observations that an electric pulse increases the medium's electric conductance. Results IRE simulation in prostate-resembling tissue shows thermal lesions with 67–92°C temperatures, which match the positions of the coagulative necrotic lesions seen in an experimental study. Simulation of IRE around a blood vessel when blood flow removes the heated blood between pulses confirms clinical observations that the perivascular tissue is thermally injured without affecting vascular patency. Conclusions The demonstration that significant Joule heating surrounds current multiple-pulsed IRE practice may contribute to future in-depth discussions on this thermal issue. This is an important subject because it has long been under-exposed in literature. Its awareness pleads for preventing IRE from calling “non-thermal” in future publications, in order to provide IRE-users with the most accurate information possible. The prospect of thermal treatment planning as outlined in this paper likely aids to the important further successful dissemination of IRE in interventional medicine. Prostate 75:332–335, 2015. © 2014 The Authors. The Prostate Published by Wiley Periodicals, Inc. PMID:25327875

Co-delivery of PSA and PSMA DNA vaccines with electroporation induces potent immune responses.

PubMed

Ferraro, Bernadette; Cisper, Neil J; Talbott, Kendra T; Philipson-Weiner, Lindsey; Lucke, Colleen E; Khan, Amir S; Sardesai, Niranjan Y; Weiner, David B

2011-01-01

Prostate cancer (PCa) remains a significant public health problem. Current treatment modalities for PCa can be useful, but may be accompanied by deleterious side effects and often do not confer long-term control. Accordingly, additional modalities, such as immunotherapy, may represent an important approach for PCa treatment. The identification of tissue-specific antigens engenders PCa an attractive target for immunotherapeutic approaches. Delivery of DNA vaccines with electroporation has shown promising results for prophylactic and therapeutic targets in a variety of species including humans. Application of this technology for PCa immunotherapy strategies has been limited to single antigen and epitope targets. We sought to test the hypothesis that a broader collection of antigens would improve the breadth and effectiveness of a PCa immune therapy approach. We therefore developed highly optimized DNA vaccines encoding prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) as a dual antigen approach to immune therapy of PCa. PSA-and PSMA-specific cellular immunogenicity was evaluated in a mouse model for co-delivery and single antigen vaccination. Mice received 2 immunizations spaced 2 weeks apart and immunogenicity was evaluated 1 week after the second vaccination. Both the PSA and PSMA vaccines induced robust antigen-specific IFNγ responses by ELISpot. Further characterization of cellular immunogenicity by flow cytometry indicated strong antigen-specific TNFα production by CD4+ T cells and IFNγ and IL-2 secretion by both CD4+ and CD8+ T cells. There was also a strong humoral response as determined by PSA-specific seroconversion. These data support further study of this novel approach to immune therapy of PCa.

KGFR as a possible therapeutic target in middle ear cholesteatoma.

PubMed

Yamamoto-Fukuda, Tomomi; Akiyama, Naotaro; Shibata, Yasuaki; Takahashi, Haruo; Ikeda, Tohru; Kohno, Michiaki; Koji, Takehiko

2014-11-01

We demonstrated that repression of keratinocyte growth factor (KGF) receptor (KGFR) could be a potentially useful strategy in the conservative treatment of middle ear cholesteatoma. Recently, the use of a selective inhibitor of the KGFR, SU5402, in an in vitro experiment resulted in the inhibition of the differentiation and proliferation of epithelial cells through KGF secretion by fibroblasts isolated from the cholesteatoma. In this study, we investigated the effects of the KGFR inhibitor during middle ear cholesteatoma formation in vivo. Based on the role of KGF in the development of cholesteatoma, Flag-hKGF cDNA driven by CMV14 promoter was transfected through electroporation into the external auditory canal of rats five times on every fourth day. Ears transfected with empty vector were used as controls. KGFR selective inhibitor (SU5402) or MEK inhibitor (PD0325901) was administered in the right ear of five rats after vector transfection. In the control, 2% DMSO in PBS was administered in the other ears after vector transfection. The use of a selective KGFR inhibitor, SU5402, completely prevented middle ear cholesteatoma formation in the rats.

Delineating the cell death mechanisms associated with skin electroporation.

PubMed

Schultheis, Katherine; Smith, Trevor R F; Kiosses, William B; Kraynyak, Kimberly A; Wong, Amelia; Oh, Janet; Broderick, Kate Elizabeth

2018-06-28

The immune responses elicited following delivery of DNA vaccines to the skin has previously been shown to be significantly enhanced by the addition of electroporation (EP) to the treatment protocol. Principally, EP increases the transfection of pDNA into the resident skin cells. In addition to increasing the levels of in vivo transfection, the physical insult induced by EP is associated with activation of innate pathways which are believed to mediate an adjuvant effect, further enhancing DNA vaccine responses. Here, we have investigated the possible mechanisms associated with this adjuvant effect, primarily focusing on the cell death pathways associated with the skin EP procedure independent of pDNA delivery. Using the minimally invasive CELLECTRA®-3P intradermal electroporation device that penetrates the epidermal and dermal layers of the skin, we have investigated apoptotic and necrotic cell death in relation to the vicinity of the electrode needles and electric field generated. Employing the well-established TUNEL assay, we detected apoptosis beginning as early as one hour after EP and peaking at the 4 hour time point. The majority of the apoptotic events were detected in the epidermal region directly adjacent to the electrode needle. Using a novel propidium iodide in vivo necrotic cell death assay, we detected necrotic events concentrated in the epidermal region adjacent to the electrode. Furthermore, we detected up-regulation of calreticulin expression on skin cells after EP, thus labeling these cells for uptake by dendritic cells and macrophages. These results allow us to delineate the cell death mechanisms occurring in the skin following intradermal EP independently of pDNA delivery. We believe these events contribute to the adjuvant effect observed following electroporation at the skin treatment site.

Electric pulses used in electrochemotherapy and electrogene therapy do not significantly change the expression profile of genes involved in the development of cancer in malignant melanoma cells.

PubMed

Mlakar, Vid; Todorovic, Vesna; Cemazar, Maja; Glavac, Damjan; Sersa, Gregor

2009-08-26

Electroporation is a versatile method for in vitro or in vivo delivery of different molecules into cells. However, no study so far has analysed the effects of electric pulses used in electrochemotherapy (ECT pulses) or electric pulses used in electrogene therapy (EGT pulses) on malignant cells. We studied the effect of ECT and EGT pulses on human malignant melanoma cells in vitro in order to understand and predict the possible effect of electric pulses on gene expression and their possible effect on cell behaviour. We used microarrays with 2698 different oligonucleotides to obtain the expression profile of genes involved in apoptosis and cancer development in a malignant melanoma cell line (SK-MEL28) exposed to ECT pulses and EGT pulses. Cells exposed to ECT pulses showed a 68.8% average survival rate, while cells exposed to EGT pulses showed a 31.4% average survival rate. Only seven common genes were found differentially expressed in cells 16 h after exposure to ECT and EGT pulses. We found that ECT and EGT pulses induce an HSP70 stress response mechanism, repress histone protein H4, a major protein involved in chromatin assembly, and down-regulate components involved in protein synthesis. Our results show that electroporation does not significantly change the expression profile of major tumour suppressor genes or oncogenes of the cell cycle. Moreover, electroporation also does not changes the expression of genes involved in the stability of DNA, supporting current evidence that electroporation is a safe method that does not promote tumorigenesis. However, in spite of being considered an isothermal method, it does to some extent induce stress, which resulted in the expression of the environmental stress response mechanism, HSP70.

Safety and Immunogenicity of PENNVAX-G DNA Prime Administered by Biojector 2000 or CELLECTRA Electroporation Device With Modified Vaccinia Ankara-CMDR Boost.

PubMed

Ake, Julie A; Schuetz, Alexandra; Pegu, Poonam; Wieczorek, Lindsay; Eller, Michael A; Kibuuka, Hannah; Sawe, Fredrick; Maboko, Leonard; Polonis, Victoria; Karasavva, Nicos; Weiner, David; Sekiziyivu, Arthur; Kosgei, Josphat; Missanga, Marco; Kroidl, Arne; Mann, Philipp; Ratto-Kim, Silvia; Anne Eller, Leigh; Earl, Patricia; Moss, Bernard; Dorsey-Spitz, Julie; Milazzo, Mark; Laissa Ouedraogo, G; Rizvi, Farrukh; Yan, Jian; Khan, Amir S; Peel, Sheila; Sardesai, Niranjan Y; Michael, Nelson L; Ngauy, Viseth; Marovich, Mary; Robb, Merlin L

2017-11-27

We report the first-in-human safety and immunogenicity evaluation of PENNVAX-G DNA/modified vaccinia Ankara-Chiang Mai double recombinant (MVA-CMDR) prime-boost human immuonodeficiency virus (HIV) vaccine, with intramuscular DNA delivery by either Biojector 2000 needle-free injection system (Biojector) or CELLECTRA electroporation device. Healthy, HIV-uninfected adults were randomized to receive 4 mg of PENNVAX-G DNA delivered intramuscularly by Biojector or electroporation at baseline and week 4 followed by intramuscular injection of 108 plaque forming units of MVA-CMDR at weeks 12 and 24. The open-label part A was conducted in the United States, followed by a double-blind, placebo-controlled part B in East Africa. Solicited and unsolicited adverse events were recorded, and immune responses were measured. Eighty-eight of 100 enrolled participants completed all study injections, which were generally safe and well tolerated, with more immediate, but transient, pain in the electroporation group. Cellular responses were observed in 57% of vaccine recipients tested and were CD4 predominant. High rates of binding antibody responses to CRF01_AE antigens, including gp70 V1V2 scaffold, were observed. Neutralizing antibodies were detected in a peripheral blood mononuclear cell assay, and moderate antibody-dependent, cell-mediated cytotoxicity activity was demonstrated. The PVG/MVA-CMDR HIV-1 vaccine regimen is safe and immunogenic. Substantial differences in safety or immunogenicity between modes of DNA delivery were not observed. NCT01260727. Published by Oxford University Press for the Infectious Diseases Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Superior induction of T cell responses to conserved HIV-1 regions by electroporated alphavirus replicon DNA compared to that with conventional plasmid DNA vaccine.

PubMed

Knudsen, Maria L; Mbewe-Mvula, Alice; Rosario, Maximillian; Johansson, Daniel X; Kakoulidou, Maria; Bridgeman, Anne; Reyes-Sandoval, Arturo; Nicosia, Alfredo; Ljungberg, Karl; Hanke, Tomás; Liljeström, Peter

2012-04-01

Vaccination using "naked" DNA is a highly attractive strategy for induction of pathogen-specific immune responses; however, it has been only weakly immunogenic in humans. Previously, we constructed DNA-launched Semliki Forest virus replicons (DREP), which stimulate pattern recognition receptors and induce augmented immune responses. Also, in vivo electroporation was shown to enhance immune responses induced by conventional DNA vaccines. Here, we combine these two approaches and show that in vivo electroporation increases CD8(+) T cell responses induced by DREP and consequently decreases the DNA dose required to induce a response. The vaccines used in this study encode the multiclade HIV-1 T cell immunogen HIVconsv, which is currently being evaluated in clinical trials. Using intradermal delivery followed by electroporation, the DREP.HIVconsv DNA dose could be reduced to as low as 3.2 ng to elicit frequencies of HIV-1-specific CD8(+) T cells comparable to those induced by 1 μg of a conventional pTH.HIVconsv DNA vaccine, representing a 625-fold molar reduction in dose. Responses induced by both DREP.HIVconsv and pTH.HIVconsv were further increased by heterologous vaccine boosts employing modified vaccinia virus Ankara MVA.HIVconsv and attenuated chimpanzee adenovirus ChAdV63.HIVconsv. Using the same HIVconsv vaccines, the mouse observations were supported by an at least 20-fold-lower dose of DNA vaccine in rhesus macaques. These data point toward a strategy for overcoming the low immunogenicity of DNA vaccines in humans and strongly support further development of the DREP vaccine platform for clinical evaluation.

Electroporation of mRNA as Universal Technology Platform to Transfect a Variety of Primary Cells with Antigens and Functional Proteins.

PubMed

Gerer, Kerstin F; Hoyer, Stefanie; Dörrie, Jan; Schaft, Niels

2017-01-01

Electroporation (EP) of mRNA into human cells is a broadly applicable method to transiently express proteins of choice in a variety of different cell types. We have spent more than a decade to optimize and adapt this method, first for antigen-loading of dendritic cells (DCs), and subsequently for T cells, B cells, bulk PBMCs, and several cell lines. In this regard, antigens were introduced, processed, and presented in context of MHC class I and II. Next to that, functional proteins like adhesion receptors, T-cell receptors (TCRs), chimeric antigen receptors (CARs), constitutively active signal transducers, and others were successfully expressed. We have also established this protocol under full GMP compliance as part of a manufacturing license to produce mRNA-electroporated DCs for therapeutic vaccination in clinical trials. Therefore, we here want to share our universal mRNA electroporation protocol and the experience we have gathered with this method. The advantages of the transfection method presented here are: (1) easy adaptation to different cell types, (2) scalability from 10 6 to approximately 10 8 cells per shot, (3) high transfection efficiency (80-99 %), (4) homogenous protein expression, (5) GMP compliance if the EP is performed in a class A clean room, and (6) no transgene integration into the genome. The provided protocol involves: Opti-MEM® as EP medium, a square-wave pulse with 500 V, and 4 mm cuvettes. To adapt the protocol to differently sized cells, simply the pulse time is altered. Next to the basic protocol, we also provide an extensive list of hints and tricks, which in our opinion are of great value for everyone who intends to use this transfection technique.

Electroporation driven delivery of both an IL-12 expressing plasmid and cisplatin synergizes to inhibit B16 melanoma tumor growth through an NK cell mediated tumor killing mechanism.

PubMed

Kim, Ha; Sin, Jeong-Im

2012-11-01

Combined therapy using chemotherapeutic drugs and immunotherapeutics offers some promise for treating patients with cancer. In this study, we evaluated whether cisplatin delivered by intratumoral (IT)-electroporation (EP) might enhance antitumor activity against established B16 melanoma and whether further addition of intramuscular (IM)-EP of IL-12 cDNA to IT-EP of cisplatin might augment antitumor therapeutic activity, with a focus on the underlining antitumor mechanism(s). When tumor (7 mm)-bearing animals were treated locally with cisplatin by IT-EP, they showed tumor growth inhibition significantly more than those without IT-EP. Moreover, IL-12 cDNA delivered by IM-EP was also able to inhibit tumor growth significantly more than control vector delivery. This tumor growth inhibition was mediated by NK cells, but not CD4+ T or CD8+ T cells, as determined by immune cell subset depletion and IFN-γ induction. Moreover, concurrent therapy using IT-EP of cisplatin plus IM-EP of IL-12 cDNA displayed antitumor therapeutic synergy. This therapeutic synergy appeared to be mediated by increased sensitivity of cisplatin-treated tumors to NK cell-mediated tumor killing. Taken together, these data support that cisplatin delivery by IT-EP plus IL-12 gene delivery by IM-EP are more effective at inducing antitumor therapeutic responses through increased sensitivity of cisplatin-treated tumors to NK cell-mediated tumor killing. This combined approach might have some implication for treating melanoma in patients.

Construction of insertion and deletion mxa mutants of Methylobacterium extorquens AM1 by electroporation.

PubMed

Toyama, H; Anthony, C; Lidstrom, M E

1998-09-01

Methylobacterium extorquens AM1 is a pink-pigmented facultative methylotroph which is widely used for analyzing pathways of C1 metabolism with biochemical and molecular biological techniques. To facilitate this approach, we have applied a new method to construct insertion or disruption mutants with drug resistance genes by electroporation. By using this method, mutants were obtained in four genes present in the mxa methylotrophy gene cluster for which the functions were unknown, mxaR, mxaS, mxaC and mxaD. These mutants were unable to grow on methanol except the mutant of mxaD, which showed reduced growth on methanol.

Electrotransfer of the epinecidin-1 gene into skeletal muscle enhances the antibacterial and immunomodulatory functions of a marine fish, grouper (Epinephelus coioides).

PubMed

Lee, Lin-Han; Hui, Cho-Fat; Chuang, Chi-Mu; Chen, Jyh-Yih

2013-11-01

Electrotransfer of plasmid DNA into skeletal muscle is a common non-viral delivery system for the study of gene function and for gene therapy. However, the effects of epinecidin-1 (epi) on bacterial growth and immune system modulation following its electrotransfer into the muscle of grouper (Epinephelus coioides), a marine fish species, have not been addressed. In this study, pCMV-gfp-epi plasmid was electroporated into grouper muscle, and its effect on subsequent infection with Vibrio vulnificus was examined. Over-expression of epi efficiently reduced bacterial numbers at 24 and 48 h after infection, and augmented the expression of immune-related genes in muscle and liver, inducing a moderate innate immune response associated with pro-inflammatory infiltration. Furthermore, electroporation of pCMV-gfp-epi plasmid without V. vulnificus infection induced moderate expression of certain immune-related genes, particularly innate immune genes. These data suggest that electroporation-mediated gene transfer of epi into the muscle of grouper may hold potential as an antimicrobial therapy for pathogen infection in marine fish. Copyright © 2013 Elsevier Ltd. All rights reserved.

Efficient mRNA-Based Genetic Engineering of Human NK Cells with High-Affinity CD16 and CCR7 Augments Rituximab-Induced ADCC against Lymphoma and Targets NK Cell Migration toward the Lymph Node-Associated Chemokine CCL19.

PubMed

Carlsten, Mattias; Levy, Emily; Karambelkar, Amrita; Li, Linhong; Reger, Robert; Berg, Maria; Peshwa, Madhusudan V; Childs, Richard W

2016-01-01

For more than a decade, investigators have pursued methods to genetically engineer natural killer (NK) cells for use in clinical therapy against cancer. Despite considerable advances in viral transduction of hematopoietic stem cells and T cells, transduction efficiencies for NK cells have remained disappointingly low. Here, we show that NK cells can be genetically reprogramed efficiently using a cGMP-compliant mRNA electroporation method that induces rapid and reproducible transgene expression in nearly all transfected cells, without negatively influencing their viability, phenotype, and cytotoxic function. To study its potential therapeutic application, we used this approach to improve key aspects involved in efficient lymphoma targeting by adoptively infused ex vivo-expanded NK cells. Electroporation of NK cells with mRNA coding for the chemokine receptor CCR7 significantly promoted migration toward the lymph node-associated chemokine CCL19. Further, introduction of mRNA coding for the high-affinity antibody-binding receptor CD16 (CD16-158V) substantially augmented NK cell cytotoxicity against rituximab-coated lymphoma cells. Based on these data, we conclude that this approach can be utilized to genetically modify multiple modalities of NK cells in a highly efficient manner with the potential to improve multiple facets of their in vivo tumor targeting, thus, opening a new arena for the development of more efficacious adoptive NK cell-based cancer immunotherapies.

Bioactivity of electric field-pulsed human recombinant interleukin-2 and its encapsulation into erythrocyte carriers.

PubMed

Mitchell, D H; James, G T; Kruse, C A

1990-06-01

The molecular integrity of human recombinant interleukin-2 (rIL-2), as measured by size exclusion chromatography, was not altered when exposed to high electrical field intensities. In addition, the biological activity was unaffected, as evidenced by the ability of the rIL-2 to stimulate the proliferation (by cell growth assays and tritiated thymidine uptake) and differentiation (by cytotoxicity assay) of human lymphocytes into killer cells. Electroporation conditions chosen for the loading of rIL-2, based upon those which provided for good recovery of carriers and minimal hemoglobin release, involved a lower field intensity (i.e., 6 kV/cm instead of 7 or 8 kV/cm) and multiple pulses (eight pulses, 5 microseconds) rather than a single pulse (40 microseconds). Human erythrocyte carriers consistently encapsulated 5-7.5% of the rIL-2 by electroporation (6 kV/cm, eight pulses, 5 microseconds duration). A rIL-2 concentration of 600,000 U/ml surrounding the erythrocytes during loading resulted in ca. 245,000 U/ml carriers, which represents a therapeutically significant quantity. Thus, rIL-2 shows potential as an encapsulated agent for slow release in the erythrocyte carrier system.

Non-thermal irreversible electroporation (N-TIRE) and adjuvant fractionated radiotherapeutic multimodal therapy for intracranial malignant glioma in a canine patient.

PubMed

Garcia, P A; Pancotto, T; Rossmeisl, J H; Henao-Guerrero, N; Gustafson, N R; Daniel, G B; Robertson, J L; Ellis, T L; Davalos, R V

2011-02-01

Non-thermal irreversible electroporation (N-TIRE) has shown promise as an ablative therapy for a variety of soft-tissue neoplasms. Here we describe the therapeutic planning aspects and first clinical application of N-TIRE for the treatment of an inoperable, spontaneous malignant intracranial glioma in a canine patient. The N-TIRE ablation was performed safely, effectively reduced the tumor volume and associated intracranial hypertension, and provided sufficient improvement in neurological function of the patient to safely undergo adjunctive fractionated radiotherapy (RT) according to current standards of care. Complete remission was achieved based on serial magnetic resonance imaging examinations of the brain, although progressive radiation encephalopathy resulted in the death of the dog 149 days after N-TIRE therapy. The length of survival of this patient was comparable to dogs with intracranial tumors treated via standard excisional surgery and adjunctive fractionated external beam RT. Our results illustrate the potential benefits of N-TIRE for in vivo ablation of undesirable brain tissue, especially when traditional methods of cytoreductive surgery are not possible or ideal, and highlight the potential radiosensitizing effects of N-TIRE on the brain.

Non-Thermal Irreversible Electroporation (N-TIRE) and Adjuvant Fractionated Radiotherapeutic Multimodal Therapy for Intracranial Malignant Glioma in a Canine Patient

PubMed Central

Garcia, P. A.; Pancotto, T.; Rossmeisl, J. H.; Henao-Guerrero, N.; Gustafson, N. R.; Daniel, G. B.; Robertson, J. L.; Ellis, T. L.; Davalos, R. V.

2011-01-01

Non-thermal irreversible electroporation (N-TIRE) has shown promise as an ablative therapy for a variety of soft-tissue neoplasms. Here we describe the therapeutic planning aspects and first clinical application of N-TIRE for the treatment of an inoperable, spontaneous malignant intracranial glioma in a canine patient. The N-TIRE ablation was performed safely, effectively reduced the tumor volume and associated intracranial hypertension, and provided sufficient improvement in neurological function of the patient to safely undergo adjunctive fractionated radiotherapy (RT) according to current standards of care. Complete remission was achieved based on serial magnetic resonance imaging examinations of the brain, although progressive radiation encephalopathy resulted in the death of the dog 149 days after N-TIRE therapy. The length of survival of this patient was comparable to dogs with intracranial tumors treated via standard excisional surgery and adjunctive fractionated external beam RT. Our results illustrate the potential benefits of N-TIRE for in vivo ablation of undesirable brain tissue, especially when traditional methods of cytoreductive surgery are not possible or ideal, and highlight the potential radiosensitizing effects of N-TIRE on the brain. PMID:21214290

Site-directed mutagenesis, in vivo electroporation and mass spectrometry in search for determinants of the subcellular targeting of Rab7b paralogue in the model eukaryote Paramecium octaurelia.

PubMed

Wyroba, E; Kwaśniak, P; Miller, K; Kobyłecki, K; Osińska, M

2016-04-11

Protein products of the paralogous genes resulting from the whole genome duplication may acquire new function. The role of post-translational modifications (PTM) in proper targeting of Paramecium Rab7b paralogue - distinct from that of Rab7a directly involved in phagocytosis - was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala200 resulted in diminished incorporation of [P32] by 37.4% and of 32 [C14-]UDP-glucose by 24%, respectively, into recombinant Rab7b_200 in comparison to the non-mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells contrary to non- mutagenized recombinant Rab7b correctly incorporated in the cytostome area. We identified the peptide ion at m/z=677.63+ characteristic for the glycan group attached to Thr200 in Rab7b using nano LC-MS/MS and comparing the peptide map of this protein with that after deglycosylation with the mixture of five enzymes of different specificity. Based on the mass of this peptide ion and quantitative radioactive assays with [P32]and  [C14-]UDP- glucose, the suggested composition of the adduct attached to Thr200 might be (Hex)1(HexNAc)1(Phos)3 or (HexNAc)1 (Deoxyhexose)1 (Phos)1 (HexA)1. These data indicate that PTM of Thr200 located in the hypervariable C-region of Rab7b in Paramecium is crucial for the proper localization/function of this protein. Moreover, these proteins differ also in other PTM: the number of phosphorylated amino acids in Rab7b is much higher than in Rab7a.

Assessment of the electrochemical effects of pulsed electric fields in a biological cell suspension.

PubMed

Chafai, Djamel Eddine; Mehle, Andraž; Tilmatine, Amar; Maouche, Bachir; Miklavčič, Damijan

2015-12-01

Electroporation of cells is successfully used in biology, biotechnology and medicine. Practical problems still arise in the electroporation of cells in suspension. For example, the determination of cell electroporation is still a demanding and time-consuming task. Electric pulses also cause contamination of the solution by the metal released from the electrodes and create local enhancements of the electric field, leading to the occurrence of electrochemical reactions at the electrode/electrolyte interface. In our study, we investigated the possibility of assessing modifications to the cell environment caused by pulsed electric fields using electrochemical impedance spectroscopy. We designed an experimental protocol to elucidate the mechanism by which a pulsed electric field affects the electrode state in relation to different electrolyte conductivities at the interface. The results show that a pulsed electric field affects electrodes and its degree depends on the electrolyte conductivity. Evolution of the electrochemical reaction rate depends on the initial free charges and those generated by the pulsed electric field. In the presence of biological cells, the initial free charges in the medium are reduced. The electrical current path at low frequency is longer, i.e., conductivity is decreased, even in the presence of increased permeability of the cell membrane created by the pulsed electric field. Copyright © 2015 Elsevier B.V. All rights reserved.

Tissue heterogeneity in structure and conductivity contribute to cell survival during irreversible electroporation ablation by “electric field sinks”

PubMed Central

Golberg, Alexander; Bruinsma, Bote G.; Uygun, Basak E.; Yarmush, Martin L.

2015-01-01

Irreversible electroporation (IRE) is an emerging, minimally invasive technique for solid tumors ablation, under clinical investigation for cancer therapy. IRE affects only the cell membrane, killing cells while preserving the extracellular matrix structure. Current reports indicate tumors recurrence rate after IRE averaging 31% of the cases, of which 10% are local recurrences. The mechanisms for these recurrences are not known and new explanations for incomplete cell death are needed. Using finite elements method for electric field distribution, we show that presence of vascular structures with blood leads to the redistribution of electric fields leading to the areas with more than 60% reduced electric field strength in proximity to large blood vessels and clustered vessel structures. In an in vivo rat model of liver IRE ablation, we show that cells located in the proximity of larger vessel structures and in proximity of clustered vessel structures appear less affected by IRE ablation than cells in the tissue parenchyma or in the proximity of small, more isolated vessels. These findings suggest a role for “electric field sinks” in local tumors recurrences after IRE and emphasize the importance of the precise mapping of the targeted organ structure and conductivity for planning of electroporation procedures. PMID:25684630

Proton pumping and the internal pH of yeast cells, measured with pyranine introduced by electroporation.

PubMed Central

Peña, A; Ramírez, J; Rosas, G; Calahorra, M

1995-01-01

The internal pH of yeast cells was determined by measuring the fluorescence changes of pyranine (8-hydroxy-1,3,6-pyrene-trisulfonic acid), which was introduced into the cells by electroporation. This may be a suitable procedure for the following reasons. (i) Only minor changes in the physiological status of the cells seemed to be produced. (ii) The dye did not seem to leak at a significant rate from the cells. (iii) Different incubation conditions produced large fluorescence changes in the dye, which in general agree with present knowledge of the proton movements of the yeast cell under different conditions. (iv) Pyranine introduced by electroporation seemed to be located in the cytoplasm and to avoid the vacuole, and therefore it probably measured actual cytoplasmic pH. (v) Correction factors to obtain a more precise estimation of the internal pH are not difficult to apply, and the procedure may be useful for other yeasts and microorganisms, as well as for the introduction of other substances into cells. Values for the cytoplasmic pHs of yeast cells that were higher than those reported previously were obtained, probably because this fluorescent indicator did not seem to penetrate into the cell vacuole. PMID:7860582

Tissue heterogeneity in structure and conductivity contribute to cell survival during irreversible electroporation ablation by "electric field sinks".

PubMed

Golberg, Alexander; Bruinsma, Bote G; Uygun, Basak E; Yarmush, Martin L

2015-02-16

Irreversible electroporation (IRE) is an emerging, minimally invasive technique for solid tumors ablation, under clinical investigation for cancer therapy. IRE affects only the cell membrane, killing cells while preserving the extracellular matrix structure. Current reports indicate tumors recurrence rate after IRE averaging 31% of the cases, of which 10% are local recurrences. The mechanisms for these recurrences are not known and new explanations for incomplete cell death are needed. Using finite elements method for electric field distribution, we show that presence of vascular structures with blood leads to the redistribution of electric fields leading to the areas with more than 60% reduced electric field strength in proximity to large blood vessels and clustered vessel structures. In an in vivo rat model of liver IRE ablation, we show that cells located in the proximity of larger vessel structures and in proximity of clustered vessel structures appear less affected by IRE ablation than cells in the tissue parenchyma or in the proximity of small, more isolated vessels. These findings suggest a role for "electric field sinks" in local tumors recurrences after IRE and emphasize the importance of the precise mapping of the targeted organ structure and conductivity for planning of electroporation procedures.

Articular chondrocyte network mediated by gap junctions: role in metabolic cartilage homeostasis.

PubMed

Mayan, Maria D; Gago-Fuentes, Raquel; Carpintero-Fernandez, Paula; Fernandez-Puente, Patricia; Filgueira-Fernandez, Purificacion; Goyanes, Noa; Valiunas, Virginijus; Brink, Peter R; Goldberg, Gary S; Blanco, Francisco J

2015-01-01

This study investigated whether chondrocytes within the cartilage matrix have the capacity to communicate through intercellular connections mediated by voltage-gated gap junction (GJ) channels. Frozen cartilage samples were used for immunofluorescence and immunohistochemistry assays. Samples were embedded in cacodylate buffer before dehydration for scanning electron microscopy. Co-immunoprecipitation experiments and mass spectrometry (MS) were performed to identify proteins that interact with the C-terminal end of Cx43. GJ communication was studied through in situ electroporation, electrophysiology and dye injection experiments. A transwell layered culture system and MS were used to identify and quantify transferred amino acids. Microscopic images revealed the presence of multiple cellular projections connecting chondrocytes within the matrix. These projections were between 5 and 150 µm in length. MS data analysis indicated that the C-terminus of Cx43 interacts with several cytoskeletal proteins implicated in Cx trafficking and GJ assembly, including α-tubulin and β-tubulin, actin, and vinculin. Electrophysiology experiments demonstrated that 12-mer oligonucleotides could be transferred between chondrocytes within 12 min after injection. Glucose was homogeneously distributed within 22 and 35 min. No transfer was detected when glucose was electroporated into A549 cells, which have no GJs. Transwell layered culture systems coupled with MS analysis revealed connexins can mediate the transfer of L-lysine and L-arginine between chondrocytes. This study reveals that intercellular connections between chondrocytes contain GJs that play a key role in cell-cell communication and a metabolic function by exchange of nutrients including glucose and essential amino acids. A three-dimensional cellular network mediated through GJs might mediate metabolic and physiological homeostasis to maintain cartilage tissue. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

A regulatory sequence from the retinoid X receptor γ gene directs expression to horizontal cells and photoreceptors in the embryonic chicken retina.

PubMed

Blixt, Maria K E; Hallböök, Finn

2016-01-01

Combining techniques of episomal vector gene-specific Cre expression and genomic integration using the piggyBac transposon system enables studies of gene expression-specific cell lineage tracing in the chicken retina. In this work, we aimed to target the retinal horizontal cell progenitors. A 208 bp gene regulatory sequence from the chicken retinoid X receptor γ gene (RXRγ208) was used to drive Cre expression. RXRγ is expressed in progenitors and photoreceptors during development. The vector was combined with a piggyBac "donor" vector containing a floxed STOP sequence followed by enhanced green fluorescent protein (EGFP), as well as a piggyBac helper vector for efficient integration into the host cell genome. The vectors were introduced into the embryonic chicken retina with in ovo electroporation. Tissue electroporation targets specific developmental time points and in specific structures. Cells that drove Cre expression from the regulatory RXRγ208 sequence excised the floxed STOP-sequence and expressed GFP. The approach generated a stable lineage with robust expression of GFP in retinal cells that have activated transcription from the RXRγ208 sequence. Furthermore, GFP was expressed in cells that express horizontal or photoreceptor markers when electroporation was performed between developmental stages 22 and 28. Electroporation of a stage 12 optic cup gave multiple cell types in accordance with RXRγ gene expression in the early retina. In this study, we describe an easy, cost-effective, and time-efficient method for testing regulatory sequences in general. More specifically, our results open up the possibility for further studies of the RXRγ-gene regulatory network governing the formation of photoreceptor and horizontal cells. In addition, the method presents approaches to target the expression of effector genes, such as regulators of cell fate or cell cycle progression, to these cells and their progenitor.

Electric pulses used in electrochemotherapy and electrogene therapy do not significantly change the expression profile of genes involved in the development of cancer in malignant melanoma cells

PubMed Central

2009-01-01

Background Electroporation is a versatile method for in vitro or in vivo delivery of different molecules into cells. However, no study so far has analysed the effects of electric pulses used in electrochemotherapy (ECT pulses) or electric pulses used in electrogene therapy (EGT pulses) on malignant cells. We studied the effect of ECT and EGT pulses on human malignant melanoma cells in vitro in order to understand and predict the possible effect of electric pulses on gene expression and their possible effect on cell behaviour. Methods We used microarrays with 2698 different oligonucleotides to obtain the expression profile of genes involved in apoptosis and cancer development in a malignant melanoma cell line (SK-MEL28) exposed to ECT pulses and EGT pulses. Results Cells exposed to ECT pulses showed a 68.8% average survival rate, while cells exposed to EGT pulses showed a 31.4% average survival rate. Only seven common genes were found differentially expressed in cells 16 h after exposure to ECT and EGT pulses. We found that ECT and EGT pulses induce an HSP70 stress response mechanism, repress histone protein H4, a major protein involved in chromatin assembly, and down-regulate components involved in protein synthesis. Conclusion Our results show that electroporation does not significantly change the expression profile of major tumour suppressor genes or oncogenes of the cell cycle. Moreover, electroporation also does not changes the expression of genes involved in the stability of DNA, supporting current evidence that electroporation is a safe method that does not promote tumorigenesis. However, in spite of being considered an isothermal method, it does to some extent induce stress, which resulted in the expression of the environmental stress response mechanism, HSP70. PMID:19709437

Cationic peptide exposure enhances pulsed-electric-field-mediated membrane disruption.

PubMed

Kennedy, Stephen M; Aiken, Erik J; Beres, Kaytlyn A; Hahn, Adam R; Kamin, Samantha J; Hagness, Susan C; Booske, John H; Murphy, William L

2014-01-01

The use of pulsed electric fields (PEFs) to irreversibly electroporate cells is a promising approach for destroying undesirable cells. This approach may gain enhanced applicability if the intensity of the PEF required to electrically disrupt cell membranes can be reduced via exposure to a molecular deliverable. This will be particularly impactful if that reduced PEF minimally influences cells that are not exposed to the deliverable. We hypothesized that the introduction of charged molecules to the cell surfaces would create regions of enhanced transmembrane electric potential in the vicinity of each charged molecule, thereby lowering the PEF intensity required to disrupt the plasma membranes. This study will therefore examine if exposure to cationic peptides can enhance a PEF's ability to disrupt plasma membranes. We exposed leukemia cells to 40 μs PEFs in media containing varying concentrations of a cationic peptide, polyarginine. We observed the internalization of a membrane integrity indicator, propidium iodide (PI), in real time. Based on an individual cell's PI fluorescence versus time signature, we were able to determine the relative degree of membrane disruption. When using 1-2 kV/cm, exposure to >50 μg/ml of polyarginine resulted in immediate and high levels of PI uptake, indicating severe membrane disruption, whereas in the absence of peptide, cells predominantly exhibited signatures indicative of no membrane disruption. Additionally, PI entered cells through the anode-facing membrane when exposed to cationic peptide, which was theoretically expected. Exposure to cationic peptides reduced the PEF intensity required to induce rapid and irreversible membrane disruption. Critically, peptide exposure reduced the PEF intensities required to elicit irreversible membrane disruption at normally sub-electroporation intensities. We believe that these cationic peptides, when coupled with current advancements in cell targeting techniques will be useful tools in applications where targeted destruction of unwanted cell populations is desired.

Cationic Peptide Exposure Enhances Pulsed-Electric-Field-Mediated Membrane Disruption

PubMed Central

Kennedy, Stephen M.; Aiken, Erik J.; Beres, Kaytlyn A.; Hahn, Adam R.; Kamin, Samantha J.; Hagness, Susan C.; Booske, John H.; Murphy, William L.

2014-01-01

Background The use of pulsed electric fields (PEFs) to irreversibly electroporate cells is a promising approach for destroying undesirable cells. This approach may gain enhanced applicability if the intensity of the PEF required to electrically disrupt cell membranes can be reduced via exposure to a molecular deliverable. This will be particularly impactful if that reduced PEF minimally influences cells that are not exposed to the deliverable. We hypothesized that the introduction of charged molecules to the cell surfaces would create regions of enhanced transmembrane electric potential in the vicinity of each charged molecule, thereby lowering the PEF intensity required to disrupt the plasma membranes. This study will therefore examine if exposure to cationic peptides can enhance a PEF’s ability to disrupt plasma membranes. Methodology/Principal Findings We exposed leukemia cells to 40 μs PEFs in media containing varying concentrations of a cationic peptide, polyarginine. We observed the internalization of a membrane integrity indicator, propidium iodide (PI), in real time. Based on an individual cell’s PI fluorescence versus time signature, we were able to determine the relative degree of membrane disruption. When using 1–2 kV/cm, exposure to >50 μg/ml of polyarginine resulted in immediate and high levels of PI uptake, indicating severe membrane disruption, whereas in the absence of peptide, cells predominantly exhibited signatures indicative of no membrane disruption. Additionally, PI entered cells through the anode-facing membrane when exposed to cationic peptide, which was theoretically expected. Conclusions/Significance Exposure to cationic peptides reduced the PEF intensity required to induce rapid and irreversible membrane disruption. Critically, peptide exposure reduced the PEF intensities required to elicit irreversible membrane disruption at normally sub-electroporation intensities. We believe that these cationic peptides, when coupled with current advancements in cell targeting techniques will be useful tools in applications where targeted destruction of unwanted cell populations is desired. PMID:24671150

Microneedles As a Delivery System for Gene Therapy

PubMed Central

Chen, Wei; Li, Hui; Shi, De; Liu, Zhenguo; Yuan, Weien

2016-01-01

Gene delivery systems can be divided to two major types: vector-based (either viral vector or non-viral vector) and physical delivery technologies. Many physical carriers, such as electroporation, gene gun, ultrasound start to be proved to have the potential to enable gene therapy. A relatively new physical delivery technology for gene delivery consists of microneedles (MNs), which has been studied in many fields and for many molecule types and indications. Microneedles can penetrate the stratum corneum, which is the main barrier for drug delivery through the skin with ease of administration and without significant pain. Many different kinds of MNs, such as metal MNs, coated MNs, dissolving MNs have turned out to be promising in gene delivery. In this review, we discussed the potential as well as the challenges of utilizing MNs to deliver nucleic acids for gene therapy. We also proposed that a combination of MNs and other gene delivery approaches may lead to a better delivery system for gene therapy. PMID:27303298

Microelectroporation device for genomic screening

DOEpatents

Perroud, Thomas D.; Renzi, Ronald F.; Negrete, Oscar; Claudnic, Mark R.

2014-09-09

We have developed an microelectroporation device that combines microarrays of oligonucleotides, microfluidic channels, and electroporation for cell transfection and high-throughput screening applications (e.g. RNA interference screens). Microarrays allow the deposition of thousands of different oligonucleotides in microscopic spots. Microfluidic channels and microwells enable efficient loading of cells into the device and prevent cross-contamination between different oligonucleotides spots. Electroporation allows optimal transfection of nucleic acids into cells (especially hard-to-transfect cells such as primary cells) by minimizing cell death while maximizing transfection efficiency. This invention has the advantage of a higher throughput and lower cost, while preventing cross-contamination compared to conventional screening technologies. Moreover, this device does not require bulky robotic liquid handling equipment and is inherently safer given that it is a closed system.

Percutaneous Irreversible Electroporation of Locally Advanced Pancreatic Carcinoma Using the Dorsal Approach: A Case Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scheffer, Hester J., E-mail: hj.scheffer@vumc.nl; Melenhorst, Marleen C. A. M., E-mail: m.melenhorst@vumc.nl; Vogel, Jantien A., E-mail: j.a.vogel@amc.uva.nl

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,more » the dorsal approach could be considered alternatively.« less

NASA Tech Briefs, October 2005

NASA Technical Reports Server (NTRS)

2005-01-01

Topics covered include: Insect-Inspired Optical-Flow Navigation Sensors; Chemical Sensors Based on Optical Ring Resonators; A Broad-Band Phase-Contrast Wave-Front Sensor; Progress in Insect-Inspired Optical Navigation Sensors; Portable Airborne Laser System Measures Forest-Canopy Height; Deployable Wide-Aperture Array Antennas; Faster Evolution of More Multifunctional Logic Circuits; Video-Camera-Based Position-Measuring System; N-Type delta Doping of High-Purity Silicon Imaging Arrays; Avionics System Architecture Tool; Updated Chemical Kinetics and Sensitivity Analysis Code; Predicting Flutter and Forced Response in Turbomachinery; Upgrades of Two Computer Codes for Analysis of Turbomachinery; Program Facilitates CMMI Appraisals; Grid Visualization Tool; Program Computes Sound Pressures at Rocket Launches; Solar-System Ephemeris Toolbox; Data-Acquisition Software for PSP/TSP Wind-Tunnel Cameras; Corrosion-Prevention Capabilities of a Water-Borne, Silicone-Based, Primerless Coating; Sol-Gel Process for Making Pt-Ru Fuel-Cell Catalysts; Making Activated Carbon for Storing Gas; System Regulates the Water Contents of Fuel-Cell Streams; Five-Axis, Three-Magnetic-Bearing Dynamic Spin Rig; Modifications of Fabrication of Vibratory Microgyroscopes; Chamber for Growing and Observing Fungi; Electroporation System for Sterilizing Water; Thermoelectric Air/Soil Energy-Harvesting Device; Flexible Metal-Fabric Radiators; Actuated Hybrid Mirror Telescope; Optical Design of an Optical Communications Terminal; Algorithm for Identifying Erroneous Rain-Gauge Readings; Condition Assessment and End-of-Life Prediction System for Electric Machines and Their Loads; Lightweight Thermal Insulation for a Liquid-Oxygen Tank; Stellar Gyroscope for Determining Attitude of a Spacecraft; and Lifting Mechanism for the Mars Explorer Rover.

Enhanced Delivery and Potency of Self-Amplifying mRNA Vaccines by Electroporation in Situ

PubMed Central

Cu, Yen; Broderick, Kate E.; Banerjee, Kaustuv; Hickman, Julie; Otten, Gillis; Barnett, Susan; Kichaev, Gleb; Sardesai, Niranjan Y.; Ulmer, Jeffrey B.; Geall, Andrew

2013-01-01

Nucleic acid-based vaccines such as viral vectors, plasmid DNA (pDNA), and mRNA are being developed as a means to address limitations of both live-attenuated and subunit vaccines. DNA vaccines have been shown to be potent in a wide variety of animal species and several products are now licensed for commercial veterinary but not human use. Electroporation delivery technologies have been shown to improve the generation of T and B cell responses from synthetic DNA vaccines in many animal species and now in humans. However, parallel RNA approaches have lagged due to potential issues of potency and production. Many of the obstacles to mRNA vaccine development have recently been addressed, resulting in a revival in the use of non-amplifying and self-amplifying mRNA for vaccine and gene therapy applications. In this paper, we explore the utility of EP for the in vivo delivery of large, self-amplifying mRNA, as measured by reporter gene expression and immunogenicity of genes encoding HIV envelope protein. These studies demonstrated that EP delivery of self-amplifying mRNA elicited strong and broad immune responses in mice, which were comparable to those induced by EP delivery of pDNA. PMID:26344119

Description of two new plasmids isolated from Francisella philomiragia strains and construction of shuttle vectors for the study of Francisella tularensis.

PubMed

Le Pihive, E; Blaha, D; Chenavas, S; Thibault, F; Vidal, D; Valade, E

2009-11-01

Francisella tularensis is the causative agent of tularemia, a zoonotic disease often transmitted to humans by infected animals. The lack of useful specific genetic tools has long hampered the study of F. tularensis subspecies. We identified and characterized two new plasmids, pF242 and pF243, isolated from Francisella philomiragia strains ATCC 25016 and ATCC 25017, respectively. Sequence analysis revealed that pF242 and pF243 are closely related to pC194 and pFNL10 plasmids, respectively. Two generations of pF242- and pF243-based shuttle vectors, harboring several antibiotic resistance markers, were developed. We used the first generation to compare transformation efficiencies in two virulent F. tularensis subspecies. We found that electroporation was more efficient than cryotransformation: almost all vectors tested were successfully introduced by electroporation into Francisella strains with a high level of efficiency. The second generation of shuttle vectors, containing a multiple cloning site and/or gfp gene downstream of Francisella groES promotor, was used for GFP production in F. tularensis. The development of new shuttle vectors offers new perspectives in the genetic manipulation of F. tularensis, helping to elucidate the mechanisms underlying its virulence.

Toward on-chip, in-cell recordings from cultured cardiomyocytes by arrays of gold mushroom-shaped microelectrodes

PubMed Central

Fendyur, Anna; Spira, Micha E.

2012-01-01

Cardiological research greatly rely on the use of cultured primary cardiomyocytes (CMs). The prime methodology to assess CM network electrophysiology is based on the use of extracellular recordings by substrate-integrated planar Micro-Electrode Arrays (MEAs). Whereas this methodology permits simultaneous, long-term monitoring of the CM electrical activity, it limits the information to extracellular field potentials (FPs). The alternative method of intracellular action potentials (APs) recordings by sharp- or patch-microelectrodes is limited to a single cell at a time. Here, we began to merge the advantages of planar MEA and intracellular microelectrodes. To that end we cultured rat CM on micrometer size protruding gold mushroom-shaped microelectrode (gMμEs) arrays. Cultured CMs engulf the gMμE permitting FPs recordings from individual cells. Local electroporation of a CM converts the extracellular recording configuration to attenuated intracellular APs with shape and duration similar to those recorded intracellularly. The procedure enables to simultaneously record APs from an unlimited number of CMs. The electroporated membrane spontaneously recovers. This allows for repeated recordings from the same CM a number of times (>8) for over 10 days. The further development of CM-gMμE configuration opens up new venues for basic and applied biomedical research. PMID:22936913

In ovo electroporation of miRNA-based plasmids in the developing neural tube and assessment of phenotypes by DiI injection in open-book preparations.

PubMed

Wilson, Nicole H; Stoeckli, Esther T

2012-10-16

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development(1,2). These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development. Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors(3) harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript(4) (located within the 3'-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown(3). Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations(5), this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development(6). This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance, the spinal cord is removed from the embryo as an open-book preparation, fixed, and injected with DiI to enable axon pathways to be traced. The spinal cord is mounted between coverslips and visualized using confocal microscopy.

Effective Protection Induced by a Monovalent DNA Vaccine against Dengue Virus (DV) Serotype 1 and a Bivalent DNA Vaccine against DV1 and DV2 in Mice.

PubMed

Zheng, Xiaoyan; Chen, Hui; Wang, Ran; Fan, Dongying; Feng, Kaihao; Gao, Na; An, Jing

2017-01-01

Dengue virus (DV) is the causal pathogen of dengue fever, which is one of the most rapidly spread mosquito-borne disease worldwide and has become a severe public health problem. Currently, there is no specific treatment for dengue; thus, a vaccine would be an effective countermeasure to reduce the morbidity and mortality. Although, the chimeric Yellow fever dengue tetravalent vaccine has been approved in some countries, it is still necessary to develop safer, more effective, and less costly vaccines. In this study, a DNA vaccine candidate pVAX1-D1ME expressing the prME protein of DV1 was inoculated in BALB/c mice via intramuscular injection or electroporation, and the immunogenicity and protection were evaluated. Compared with traditional intramuscular injection, administration with 50 μg pVAX1-D1ME via electroporation with three immunizations induced persistent humoral and cellular immune responses and effectively protected mice against lethal DV1 challenge. In addition, immunization with a bivalent vaccine consisting of pVAX1-D1ME and pVAX1-D2ME via electroporation generated a balanced IgG response and neutralizing antibodies against DV1 and DV2 and could protect mice from lethal challenge with DV1 and DV2. This study sheds new light on developing a dengue tetravalent DNA vaccine.

Non-Viral Transfection Methods Optimized for Gene Delivery to a Lung Cancer Cell Line

PubMed Central

Salimzadeh, Loghman; Jaberipour, Mansooreh; Hosseini, Ahmad; Ghaderi, Abbas

2013-01-01

Background Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. Methods In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize delivery of a plasmid construct that expressed Green Fluorescent Protein (GFP). Transgene expression was detected by fluorescent microscopy and flowcytometry. Toxicities of the methods were estimated by trypan blue staining. In order to evaluate the density of the transfected gene, we used a plasmid construct that expressed the Stromal cell-Derived Factor-1 (SDF-1) gene and measured its expression by real-time PCR. Results Mean levels of GFP-expressing cells 48 hr after transfection were 8.4% (CaP), 8.2% (DEAE-dextran), 4.9% (superfect), 34.1% (electroporation), and 40.1% (lipofection). Lipofection had the highest intense SDF-1 expression of the analyzed methods. Conclusion This study has shown that the lipofection and electroporation methods were more efficient at gene delivery to Mehr-80 cells. The quantity of DNA per transfection, reagent concentration, and incubation time were identified as essential factors for successful transfection in all of the studied methods. PMID:23799175

Keratinocyte Growth Factor Gene Electroporation into Skeletal Muscle as a Novel Gene Therapeutic Approach for Elastase-Induced Pulmonary Emphysema in Mice.

PubMed

Tobinaga, Shuichi; Matsumoto, Keitaro; Nagayasu, Takeshi; Furukawa, Katsuro; Abo, Takafumi; Yamasaki, Naoya; Tsuchiya, Tomoshi; Miyazaki, Takuro; Koji, Takehiko

2015-06-29

Pulmonary emphysema is a progressive disease with airspace destruction and an effective therapy is needed. Keratinocyte growth factor (KGF) promotes pulmonary epithelial proliferation and has the potential to induce lung regeneration. The aim of this study was to determine the possibility of using KGF gene therapy for treatment of a mouse emphysema model induced by porcine pancreatic elastase (PPE). Eight-week-old BALB/c male mice treated with intra-tracheal PPE administration were transfected with 80 μg of a recombinant human KGF (rhKGF)-expressing FLAG-CMV14 plasmid (pKGF-FLAG gene), or with the pFLAG gene expressing plasmid as a control, into the quadriceps muscle by electroporation. In the lung, the expression of proliferating cell nuclear antigen (PCNA) was augmented, and surfactant protein A (SP-A) and KGF receptor (KGFR) were co-expressed in PCNA-positive cells. Moreover, endogenous KGF and KGFR gene expression increased significantly by pKGF-FLAG gene transfection. Arterial blood gas analysis revealed that the PaO2 level was not significantly reduced on day 14 after PPE instillation with pKGF-FLAG gene transfection compared to that of normal mice. These results indicated that KGF gene therapy with electroporation stimulated lung epithelial proliferation and protected depression of pulmonary function in a mouse emphysema model, suggesting a possible method of treating pulmonary emphysema.

Keratinocyte Growth Factor Gene Electroporation into Skeletal Muscle as a Novel Gene Therapeutic Approach for Elastase-Induced Pulmonary Emphysema in Mice

PubMed Central

Tobinaga, Shuichi; Matsumoto, Keitaro; Nagayasu, Takeshi; Furukawa, Katsuro; Abo, Takafumi; Yamasaki, Naoya; Tsuchiya, Tomoshi; Miyazaki, Takuro; Koji, Takehiko

2015-01-01

Pulmonary emphysema is a progressive disease with airspace destruction and an effective therapy is needed. Keratinocyte growth factor (KGF) promotes pulmonary epithelial proliferation and has the potential to induce lung regeneration. The aim of this study was to determine the possibility of using KGF gene therapy for treatment of a mouse emphysema model induced by porcine pancreatic elastase (PPE). Eight-week-old BALB/c male mice treated with intra-tracheal PPE administration were transfected with 80 μg of a recombinant human KGF (rhKGF)-expressing FLAG-CMV14 plasmid (pKGF-FLAG gene), or with the pFLAG gene expressing plasmid as a control, into the quadriceps muscle by electroporation. In the lung, the expression of proliferating cell nuclear antigen (PCNA) was augmented, and surfactant protein A (SP-A) and KGF receptor (KGFR) were co-expressed in PCNA-positive cells. Moreover, endogenous KGF and KGFR gene expression increased significantly by pKGF-FLAG gene transfection. Arterial blood gas analysis revealed that the PaO2 level was not significantly reduced on day 14 after PPE instillation with pKGF-FLAG gene transfection compared to that of normal mice. These results indicated that KGF gene therapy with electroporation stimulated lung epithelial proliferation and protected depression of pulmonary function in a mouse emphysema model, suggesting a possible method of treating pulmonary emphysema. PMID:26160987

Intra-operative navigation of a 3-dimensional needle localization system for precision of irreversible electroporation needles in locally advanced pancreatic cancer.

PubMed

Bond, L; Schulz, B; VanMeter, T; Martin, R C G

2017-02-01

Irreversible electroporation (IRE) uses multiple needles and a series of electrical pulses to create pores in cell membranes and cause cell apoptosis. One of the demands of IRE is the precise needle spacing required. Two-dimensional intraoperative ultrasound (2-D iUS) is currently used to measure inter-needle distances but requires significant expertise. This study evaluates the potential of three-dimensional (3-D) image guidance for placing IRE needles and calculating needle spacing. A prospective clinical evaluation of a 3-D needle localization system (Explorer™) was evaluated in consecutive patients from April 2012 through June 2013 for unresectable pancreatic adenocarcinoma. 3-D reconstructions of patients' anatomy were generated from preoperative CT images, which were aligned to the intraoperative space. Thirty consecutive patients with locally advanced pancreatic cancer were treated with IRE. The needle localization system setup added an average of 6.5 min to each procedure. The 3-D needle localization system increased surgeon confidence and ultimately reduced needle placement time. IRE treatment efficacy is highly dependent on accurate needle spacing. The needle localization system evaluated in this study aims to mitigate these issues by providing the surgeon with additional visualization and data in 3-D. The Explorer™ system provides valuable guidance information and inter-needle distance calculations. Copyright © 2016 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.

All-in-one processing of heterogeneous human cell grafts for gene and cell therapy.

PubMed

Lukianova-Hleb, Ekaterina Y; Yvon, Eric S; Shpall, Elizabeth J; Lapotko, Dmitri O

2016-01-01

Current cell processing technologies for gene and cell therapies are often slow, expensive, labor intensive and are compromised by high cell losses and poor selectivity thus limiting the efficacy and availability of clinical cell therapies. We employ cell-specific on-demand mechanical intracellular impact from laser pulse-activated plasmonic nanobubbles (PNB) to process heterogeneous human cell grafts ex vivo with dual simultaneous functionality, the high cell type specificity, efficacy and processing rate for transfection of target CD3+ cells and elimination of subsets of unwanted CD25+ cells. The developed bulk flow PNB system selectively processed human cells at a rate of up to 100 million cell/minute, providing simultaneous transfection of CD3+ cells with the therapeutic gene (FKBP12(V36)-p30Caspase9) with the efficacy of 77% and viability 95% (versus 12 and 60%, respectively, for standard electroporation) and elimination of CD25+ cells with 99% efficacy. PNB flow technology can unite and replace several methodologies in an all-in-one universal ex vivo simultaneous procedure to precisely and rapidly prepare a cell graft for therapy. PNB's can process various cell systems including cord blood, stem cells, and bone marrow.

Potentiation of an anthrax DNA vaccine with electroporation.

PubMed

Luxembourg, A; Hannaman, D; Nolan, E; Ellefsen, B; Nakamura, G; Chau, L; Tellez, O; Little, S; Bernard, R

2008-09-19

DNA vaccines are a promising method of immunization against biothreats and emerging infections because they are relatively easy to design, manufacture, store and distribute. However, immunization with DNA vaccines using conventional delivery methods often fails to induce consistent, robust immune responses, especially in species larger than the mouse. Intramuscular (i.m.) delivery of a plasmid encoding anthrax toxin protective antigen (PA) using electroporation (EP), a potent DNA delivery method, rapidly induced anti-PA IgG and toxin neutralizing antibodies within 2 weeks following a single immunization in multiple experimental species. The delivery procedure is particularly dose efficient and thus favorable for achieving target levels of response following vaccine administration in humans. These results suggest that EP may be a valuable platform technology for the delivery of DNA vaccines against anthrax and other biothreat agents.

Electric field computation and measurements in the electroporation of inhomogeneous samples

NASA Astrophysics Data System (ADS)

Bernardis, Alessia; Bullo, Marco; Campana, Luca Giovanni; Di Barba, Paolo; Dughiero, Fabrizio; Forzan, Michele; Mognaschi, Maria Evelina; Sgarbossa, Paolo; Sieni, Elisabetta

2017-12-01

In clinical treatments of a class of tumors, e.g. skin tumors, the drug uptake of tumor tissue is helped by means of a pulsed electric field, which permeabilizes the cell membranes. This technique, which is called electroporation, exploits the conductivity of the tissues: however, the tumor tissue could be characterized by inhomogeneous areas, eventually causing a non-uniform distribution of current. In this paper, the authors propose a field model to predict the effect of tissue inhomogeneity, which can affect the current density distribution. In particular, finite-element simulations, considering non-linear conductivity against field relationship, are developed. Measurements on a set of samples subject to controlled inhomogeneity make it possible to assess the numerical model in view of identifying the equivalent resistance between pairs of electrodes.

Differential effects of human papillomavirus type 6, 16, and 18 DNAs on immortalization and transformation of human cervical epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pecoraro, G.; Morgan, D.; Defendi, V.

1989-01-01

The human papillomaviruses (HPVs) are associated with specific benign and malignant lesions of the skin and mucosal epithelia. Cloned viral DNAs from HPV types 6b, 16, and 18 associated with different pathological manifestations of genital neoplasia in vivo were introduced into primary human cervical epithelial cells by electroporation. Cells transfected with HPV16 or HPV18 DNA acquired indefinite lifespans, distinct morphological alterations, and anchorage-independent growth (HPV18), and contain integrated transcriptionally active viral genomes. HPV6b or plasmid electroporated cells senesced at low passage. The alterations in growth and differentiation of the cells appear to reflect the progressive oncogenic processes that result inmore » cervical carcinoma in vivo.« less

A seepage investigation of an area at and near Oak Ridge National Laboratory, Oak Ridge, Tennessee, March through August 1993

USGS Publications Warehouse

Johnson, G.C.

1996-01-01

A seepage investigation was conducted of an area surrounding the Oak Ridge National Laboratory from March through August 1993. The project was divided into three phases: a reconnaissance to inventory and map seeps, springs, and stream-measurement sites; a high base flow seepage investigation; and a low base flow seepage investigation. The reconnaissance consisted of following each tributary to its source to inventory each site where water was issuing from the ground. Stream- measurement sites were also located along stream reaches at 500-foot intervals. A total of 822 sites were identified. A global positioning system was used to locate 483 sites to within 3- to 5-meter accuracy. The high base flow seepage investigation was conducted from April 29 through May 3, 1993, and from May 7 through May 10, 1993. During the high base flow seepage investigation, sites identified during the reconnaissance were revisited. At almost all sites with flowing water, discharge, pH, specific conductance, and temperature were recorded. Two hundred and fourteen sites were dry. The low base flow seepage investigation was conducted from August 8 through August 10, 1993, and consisted of revisiting the seeps and springs that were flowing during the high base flow seepage investigation. Stream- measurement sites were not revisited. One hundred and forty-one sites were dry.

A Novel siRNA-Based Approach to Study Mechanisms of Resistance/Action of a New Drug in Treatment of Breast Cancer. Addendum

DTIC Science & Technology

2006-08-01

electroporation, were tested in the MCF-7 breast cancer cell line. The cell line was then treated with a lethal dose of ET-743 and cytarabine , however no...drugs with known mechanisms of resistance, methotrexate (MTX) and cytarabine , using a clonogenic assay and MCF-7 breast cancer cells. 3. To employ...Aim 2. To test the ability of the generated siRNA library by using two drugs with known mechanisms of resistance, methotrexate (MTX) and cytarabine

Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with Various Plasmids

PubMed Central

Serror, Pascale; Sasaki, Takashi; Ehrlich, S. Dusko; Maguin, Emmanuelle

2002-01-01

We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 104 transformants per μg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a good basis for developing molecular tools for L. delbrueckii and open the field of genetic studies in L. delbrueckii. PMID:11772607

[Acceleration of osmotic dehydration process through ohmic heating of foods: raspberries (Rubus idaeus)].

PubMed

Simpson, Ricardo R; Jiménez, Maite P; Carevic, Erica G; Grancelli, Romina M

2007-06-01

Raspberries (Rubus idaeus) were osmotically dehydrated by applying a conventional method under the supposition of a homogeneous solution, all in a 62% glucose solution at 50 degrees C. Raspberries (Rubus idaeus) were also osmotically dehydrated by using ohmic heating in a 57% glucose solution at a variable voltage (to maintain temperature between 40 and 50 degrees C) and an electric field intensity <100 V/cm. When comparing the results from both experiments it was evident that processing time is reduced when ohmic heating technique was used. In some cases this reduction reached even 50%. This is explained by the additional effect to the thermal damage that is generated in an ohmic process, denominated electroporation.

Glutathione and fungal elicitor regulation of a plant defense gene promoter in electroporated protoplasts

PubMed Central

Dron, Michel; Clouse, Steven D.; Dixon, Richard A.; Lawton, Michael A.; Lamb, Christopher J.

1988-01-01

To investigate the mechanisms underlying activation of plant defenses against microbial attack we have studied elicitor regulation of a chimeric gene comprising the 5′ flanking region of a defense gene encoding the phytoalexin biosynthetic enzyme chalcone synthase fused to a bacterial chloramphenicol acetyltransferase gene. Glutathione or fungal elicitor caused a rapid, marked but transient expression of the chimeric gene electroporated into soybean protoplasts. The response closely resembled that of endogenous chalcone synthase genes in suspension cultured cells. Functional analysis of 5′ deletions suggests that promoter activity is determined by an elicitor-regulated activator located between the “TATA box” and nucleotide position -173 and an upstream silencer between -173 and -326. These cis-acting elements function in the transduction of the elicitation signal to initiate elaboration of an inducible defense response. Images PMID:16593981

A multiagent filovirus DNA vaccine delivered by intramuscular electroporation completely protects mice from ebola and Marburg virus challenge.

PubMed

Grant-Klein, Rebecca J; Van Deusen, Nicole M; Badger, Catherine V; Hannaman, Drew; Dupuy, Lesley C; Schmaljohn, Connie S

2012-11-01

We evaluated the immunogenicity and protective efficacy of DNA vaccines expressing the codon-optimized envelope glycoprotein genes of Zaire ebolavirus, Sudan ebolavirus, and Marburg marburgvirus (Musoke and Ravn). Intramuscular or intradermal delivery of the vaccines in BALB/c mice was performed using the TriGrid™ electroporation device. Mice that received DNA vaccines against the individual viruses developed robust glycoprotein-specific antibody titers as determined by ELISA and survived lethal viral challenge with no display of clinical signs of infection. Survival curve analysis revealed there was a statistically significant increase in survival compared to the control groups for both the Ebola and Ravn virus challenges. These data suggest that further analysis of the immune responses generated in the mice and additional protection studies in nonhuman primates are warranted.

Possible mechanisms for delayed neurological damage in lightning and electrical injury.

PubMed

Reisner, Andrew D

2013-01-01

This article provides and reviews hypotheses to help explain the poorly understood phenomenon of delayed neurological injury following lightning or electrical injury. A review of extant literature provides a starting point to integrate what is already known in an attempt to provide new hypotheses for this phenomenon, as well as to discuss existing hypotheses. The author proposes two theories which stem from the literature on the damaging effects of oxidative stress, and also reviews an existing hypothesis, the electroporation hypothesis. The former two theories can account for delayed damage which is either of vascular or nonvascular origin. The electroporation hypothesis can explain changes both in cases where there is cellular loss as well as cases where there only appears to be change in function after lightning or electrical injury. Although all theories discussed are speculative, the formation of hypotheses is always a starting point in the scientific process. In cases where there is delayed neurological damage with a vascular origin, it is possible that free radicals resulting from oxidative stress may gradually damage spinal vascular endothelial cells, cutting off blood supply, and ending in death of spinal neurons. When the delayed condition is demyelination without vascular damage, it is possible that the free radicals from oxidative stress are formed directly from the lipids found in abundance in myelin cells. The electroporation hypothesis, the formation of additional pores in neurons, may best explain immediate or progressive changes in structure and function after lightning or electrical injury.

A DNA vaccine delivered by dermal electroporation fully protects cynomolgus macaques against Lassa fever.

PubMed

Cashman, Kathleen A; Wilkinson, Eric R; Shaia, Carl I; Facemire, Paul R; Bell, Todd M; Bearss, Jeremy J; Shamblin, Joshua D; Wollen, Suzanne E; Broderick, Kate E; Sardesai, Niranjan Y; Schmaljohn, Connie S

2017-12-02

Lassa virus (LASV) is an ambisense RNA virus in the Arenaviridae family and is the etiological agent of Lassa fever, a severe hemorrhagic disease endemic to West and Central Africa. 1,2 There are no US Food and Drug Administration (FDA)-licensed vaccines available to prevent Lassa fever. 1,2 in our previous studies, we developed a gene-optimized DNA vaccine that encodes the glycoprotein precursor gene of LASV (Josiah strain) and demonstrated that 3 vaccinations accompanied by dermal electroporation protected guinea pigs from LASV-associated illness and death. Here, we describe an initial efficacy experiment in cynomolgus macaque nonhuman primates (NHPs) in which we followed an identical 3-dose vaccine schedule that was successful in guinea pigs, and a follow-on experiment in which we used an accelerated vaccination strategy consisting of 2 administrations, spaced 4 weeks apart. In both studies, all of the LASV DNA-vaccinated NHPs survived challenge and none of them had measureable, sustained viremia or displayed weight loss or other disease signs post-exposure. Three of 10 mock-vaccinates survived exposure to LASV, but all of them became acutely ill post-exposure and remained chronically ill to the study end point (45 d post-exposure). Two of the 3 survivors experienced sensorineural hearing loss (described elsewhere). These results clearly demonstrate that the LASV DNA vaccine combined with dermal electroporation is a highly effective candidate for eventual use in humans.

Asymmetric Waveforms Decrease Lethal Thresholds in High Frequency Irreversible Electroporation Therapies

PubMed Central

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. PMID:28106146

Plasma-mediated transfection of RPE

NASA Astrophysics Data System (ADS)

Palanker, D.; Chalberg, T.; Vankov, A.; Huie, P.; Molnar, F. E.; Butterwick, A.; Calos, M.; Marmor, M.; Blumenkranz, M. S.

2006-02-01

A major obstacle in applying gene therapy to clinical practice is the lack of efficient and safe gene delivery techniques. Viral delivery has encountered a number of serious problems including immunological reactions and malignancy. Non-viral delivery methods (liposomes, sonoporation and electroporation) have either low efficiency in-vivo or produce severe collateral damage to ocular tissues. We discovered that tensile stress greatly increases the susceptibility of cellular membranes to electroporation. For synchronous application of electric field and mechanical stress, both are generated by the electric discharge itself. A pressure wave is produced by rapid vaporization of the medium. To prevent termination of electric current by the vapor cavity it is ionized thus restoring its electric conductivity. For in-vivo experiments with rabbits a plasmid DNA was injected into the subretinal space, and RPE was treated trans-sclerally with an array of microelectodes placed outside the eye. Application of 250-300V and 100-200 μs biphasic pulses via a microelectrode array resulted in efficient transfection of RPE without visible damage to the retina. Gene expression was quantified and monitored using bioluminescence (luciferase) and fluorescence (GFP) imaging. Transfection efficiency of RPE with this new technique exceeded that of standard electroporation by a factor 10,000. Safe and effective non-viral DNA delivery to the mammalian retina may help to materialize the enormous potential of the ocular gene therapy. Future experiments will focus on continued characterization of the safety and efficacy of this method and evaluation of long-term transgene expression in the presence of phiC31 integrase.

Asymmetric Waveforms Decrease Lethal Thresholds in High Frequency Irreversible Electroporation Therapies

NASA Astrophysics Data System (ADS)

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.

PLK-1 Silencing in Bladder Cancer by siRNA Delivered With Exosomes.

PubMed

Greco, Kristin A; Franzen, Carrie A; Foreman, Kimberly E; Flanigan, Robert C; Kuo, Paul C; Gupta, Gopal N

2016-05-01

To use exosomes as a vector to deliver small interfering ribonucleic acid (siRNA) to silence the polo-like kinase 1 (PLK-1) gene in bladder cancer cells. Exosomes were isolated from both human embryonic kidney 293 (HEK293) cell and mesenchymal stem cell (MSC) conditioned media. Fluorescently labeled exosomes were co-cultured with bladder cancer and normal epithelial cells and uptake was quantified by image cytometry. PLK-1 siRNA and negative control siRNA were loaded into HEK293 and MSC exosomes using electroporation. An invasive bladder cancer cell line (UMUC3) was co-cultured with the electroporated exosomes. Quantitative reverse transcriptase polymerase chain reaction was performed. Protein analysis was performed by Western blot. Annexin V staining and MTT assays were used to investigate effects on apoptosis and viability. Bladder cancer cell lines internalize an increased percentage of HEK293 exosomes when compared to normal bladder epithelial cells. Treatment of UMUC3 cells with exosomes electroporated with PLK-1 siRNA achieved successful knockdown of PLK-1 mRNA and protein when compared to cells treated with negative control exosomes. HEK293 and MSC exosomes were effectively used as a delivery vector to transport PLK-1 siRNA to bladder cancer cells in vitro, resulting in selective gene silencing of PLK-1. The use of exosomes as a delivery vector for potential intravesical therapy is attractive. Copyright © 2016 Elsevier Inc. All rights reserved.

In vivo real-time monitoring system of electroporation mediated control of transdermal and topical drug delivery.

PubMed

Blagus, Tanja; Markelc, Bostjan; Cemazar, Maja; Kosjek, Tina; Preat, Veronique; Miklavcic, Damijan; Sersa, Gregor

2013-12-28

Electroporation (EP) is a physical method for the delivery of molecules into cells and tissues, including the skin. In this study, in order to control the degree of transdermal and topical drug delivery, EP at different amplitudes of electric pulses was evaluated. A new in vivo real-time monitoring system based on fluorescently labeled molecules was developed, for the quantification of transdermal and topical drug delivery. EP of the mouse skin was performed with new non-invasive multi-array electrodes, delivering different amplitudes of electric pulses ranging from 70 to 570 V, between the electrode pin pairs. Patches, soaked with 4 kDa fluorescein-isothiocyanate labeled dextran (FD), doxorubicin (DOX) or fentanyl (FEN), were applied to the skin before and after EP. The new monitoring system was developed based on the delivery of FD to and through the skin. FD relative quantity was determined with fluorescence microscopy imaging, in the treated region of the skin for topical delivery and in a segment of the mouse tail for transdermal delivery. The application of electric pulses for FD delivery resulted in enhanced transdermal delivery. Depending on the amplitude of electric pulses, it increased up to the amplitude of 360 V, and decreased at higher amplitudes (460 and 570 V). Topical delivery steadily enhanced with increasing the amplitude of the delivered electric pulses, being even higher than after tape stripping used as a positive control. The non-invasive monitoring of the delivery of DOX, a fluorescent chemotherapeutic drug, qualitatively and quantitatively confirmed the effects of EP at 360 and 570 V pulse amplitudes on topical and transdermal drug delivery. Delivery of FEN at 360 and 570 V pulse amplitudes verified the observed effects as obtained with FD and DOX, by the measured physiological responses of the mice as well as FEN plasma concentration. This study demonstrates that with the newly developed non-invasive multi-array electrodes and with the varying electric pulse amplitude, the amount of topical and transdermal drug delivery to the skin can be controlled. Furthermore, the newly developed monitoring system provides a tool for rapid real-time determination of both, transdermal and topical delivery, when the delivered molecule is fluorescent. © 2013 Elsevier B.V. All rights reserved.

Cerebroventricular Microinjection (CVMI) into Adult Zebrafish Brain Is an Efficient Misexpression Method for Forebrain Ventricular Cells

PubMed Central

Kizil, Caghan; Brand, Michael

2011-01-01

The teleost fish Danio rerio (zebrafish) has a remarkable ability to generate newborn neurons in its brain at adult stages of its lifespan-a process called adult neurogenesis. This ability relies on proliferating ventricular progenitors and is in striking contrast to mammalian brains that have rather restricted capacity for adult neurogenesis. Therefore, investigating the zebrafish brain can help not only to elucidate the molecular mechanisms of widespread adult neurogenesis in a vertebrate species, but also to design therapies in humans with what we learn from this teleost. Yet, understanding the cellular behavior and molecular programs underlying different biological processes in the adult zebrafish brain requires techniques that allow manipulation of gene function. As a complementary method to the currently used misexpression techniques in zebrafish, such as transgenic approaches or electroporation-based delivery of DNA, we devised a cerebroventricular microinjection (CVMI)-assisted knockdown protocol that relies on vivo morpholino oligonucleotides, which do not require electroporation for cellular uptake. This rapid method allows uniform and efficient knockdown of genes in the ventricular cells of the zebrafish brain, which contain the neurogenic progenitors. We also provide data on the use of CVMI for growth factor administration to the brain – in our case FGF8, which modulates the proliferation rate of the ventricular cells. In this paper, we describe the CVMI method and discuss its potential uses in zebrafish. PMID:22076157

Evaluation of the safety of irreversible electroporation on the stomach wall using a pig model

PubMed Central

Li, Jiannan; Zeng, Jianying; Chen, Jibing; Shi, Jian; Luo, Xiaomei; Fang, Gang; Chai, Wei; Zhang, Wenlong; Liu, Tongjun; Niu, Lizhi

2017-01-01

The aim of the present study was to evaluate the effects of irreversible electroporation (IRE) on the stomach wall following the direct application of IRE onto the organ surface. IRE ablation was performed in 8 Tibetan mini-pigs, which were randomly assigned into two groups based on their ablated areas: Group A, gastric cardia, fundus of stomach, gastric body and group B, lesser gastric curvature, greater gastric curvature, stomach pylorus. Two IRE needles were placed in the space between the stomach wall and the liver (not inserted into the stomach tissue), and three lesions were created in each pig. Serum aminotransferase and white blood cell (WBC) levels were measured. Gastroscopy and endoscopic ultrasonography were performed. From each group, 2 pigs were sacrificed on day 7 post-IRE; the remaining pigs were sacrificed on day 28 post-IRE. There were no signs of perforation on the stomach wall. Serum aminotransferase and WBC levels increased in both groups on day 1 post-IRE and decreased gradually thereafter. The gastroscopy procedure revealed oval ulcers on day 7 post-IRE and smaller ulcers on day 28 post-IRE. Transmural necrosis, inflammation and fibrosis were observed at 7 days post-IRE. Healing ulcers were observed at 28 days post-IRE. In conclusion, IRE ablation caused damage to the stomach wall; however, IRE did not induce any perforation. PMID:28672987

Intracellular ROS mediates gas plasma-facilitated cellular transfection in 2D and 3D cultures

PubMed Central

Xu, Dehui; Wang, Biqing; Xu, Yujing; Chen, Zeyu; Cui, Qinjie; Yang, Yanjie; Chen, Hailan; Kong, Michael G.

2016-01-01

This study reports the potential of cold atmospheric plasma (CAP) as a versatile tool for delivering oligonucleotides into mammalian cells. Compared to lipofection and electroporation methods, plasma transfection showed a better uptake efficiency and less cell death in the transfection of oligonucleotides. We demonstrated that the level of extracellular aqueous reactive oxygen species (ROS) produced by gas plasma is correlated with the uptake efficiency and that this is achieved through an increase of intracellular ROS levels and the resulting increase in cell membrane permeability. This finding was supported by the use of ROS scavengers, which reduced CAP-based uptake efficiency. In addition, we found that cold atmospheric plasma could transfer oligonucleotides such as siRNA and miRNA into cells even in 3D cultures, thus suggesting the potential for unique applications of CAP beyond those provided by standard transfection techniques. Together, our results suggest that cold plasma might provide an efficient technique for the delivery of siRNA and miRNA in 2D and 3D culture models. PMID:27296089

Methods for Engineering Sulfate Reducing Bacteria of the Genus Desulfovibrio

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chhabra, Swapnil R; Keller, Kimberly L.; Wall, Judy D.

Sulfate reducing bacteria are physiologically important given their nearly ubiquitous presence and have important applications in the areas of bioremediation and bioenergy. This chapter provides details on the steps used for homologous-recombination mediated chromosomal manipulation of Desulfovibrio vulgaris Hildenborough, a well-studied sulfate reducer. More specifically, we focus on the implementation of a 'parts' based approach for suicide vector assembly, important aspects of anaerobic culturing, choices for antibiotic selection, electroporation-based DNA transformation, as well as tools for screening and verifying genetically modified constructs. These methods, which in principle may be extended to other sulfate-reducing bacteria, are applicable for functional genomics investigations,more » as well as metabolic engineering manipulations.« less

Magneto-electric Nanoparticles to Enable Field-controlled High-Specificity Drug Delivery to Eradicate Ovarian Cancer Cells

PubMed Central

Guduru, Rakesh; Liang, Ping; Runowicz, Carolyn; Nair, Madhavan; Atluri, Venkata; Khizroev, Sakhrat

2013-01-01

The nanotechnology capable of high-specificity targeted delivery of anti-neoplastic drugs would be a significant breakthrough in Cancer in general and Ovarian Cancer in particular. We addressed this challenge through a new physical concept that exploited (i) the difference in the membrane electric properties between the tumor and healthy cells and (ii) the capability of magneto-electric nanoparticles (MENs) to serve as nanosized converters of remote magnetic field energy into the MENs' intrinsic electric field energy. This capability allows to remotely control the membrane electric fields and consequently trigger high-specificity drug uptake through creation of localized nano-electroporation sites. In in-vitro studies on human ovarian carcinoma cell (SKOV-3) and healthy cell (HOMEC) lines, we applied a 30-Oe d.c. field to trigger high-specificity uptake of paclitaxel loaded on 30-nm CoFe2O4@BaTiO3 MENs. The drug penetrated through the membrane and completely eradicated the tumor within 24 hours without affecting the normal cells. PMID:24129652

Magneto-electric nanoparticles to enable field-controlled high-specificity drug delivery to eradicate ovarian cancer cells.

PubMed

Guduru, Rakesh; Liang, Ping; Runowicz, Carolyn; Nair, Madhavan; Atluri, Venkata; Khizroev, Sakhrat

2013-10-16

The nanotechnology capable of high-specificity targeted delivery of anti-neoplastic drugs would be a significant breakthrough in Cancer in general and Ovarian Cancer in particular. We addressed this challenge through a new physical concept that exploited (i) the difference in the membrane electric properties between the tumor and healthy cells and (ii) the capability of magneto-electric nanoparticles (MENs) to serve as nanosized converters of remote magnetic field energy into the MENs' intrinsic electric field energy. This capability allows to remotely control the membrane electric fields and consequently trigger high-specificity drug uptake through creation of localized nano-electroporation sites. In in-vitro studies on human ovarian carcinoma cell (SKOV-3) and healthy cell (HOMEC) lines, we applied a 30-Oe d.c. field to trigger high-specificity uptake of paclitaxel loaded on 30-nm CoFe₂O₄ @BaTiO₃ MENs. The drug penetrated through the membrane and completely eradicated the tumor within 24 hours without affecting the normal cells.

Follistatin induces muscle hypertrophy through satellite cell proliferation and inhibition of both myostatin and activin.

PubMed

Gilson, Hélène; Schakman, Olivier; Kalista, Stéphanie; Lause, Pascale; Tsuchida, Kunihiro; Thissen, Jean-Paul

2009-07-01

Follistatin (FS) inhibits several members of the TGF-beta superfamily, including myostatin (Mstn), a negative regulator of muscle growth. Mstn inhibition by FS represents a potential therapeutic approach of muscle atrophy. The aim of our study was to investigate the mechanisms of the FS-induced muscle hypertrophy. To test the role of satellite cells in the FS effect, we used irradiation to destroy their proliferative capacity. FS overexpression increased the muscle weight by about 37% in control animals, but the increase reached only 20% in irradiated muscle, supporting the role of cell proliferation in the FS-induced hypertrophy. Surprisingly, the muscle hypertrophy caused by FS reached the same magnitude in Mstn-KO as in WT mice, suggesting that Mstn might not be the only ligand of FS involved in the regulation of muscle mass. To assess the role of activin (Act), another FS ligand, in the FS-induced hypertrophy, we electroporated FSI-I, a FS mutant that does not bind Act with high affinity. Whereas FS electroporation increased muscle weight by 32%, the muscle weight gain induced by FSI-I reached only 14%. Furthermore, in Mstn-KO mice, FSI-I overexpression failed to induce hypertrophy, in contrast to FS. Therefore, these results suggest that Act inhibition may contribute to FS-induced hypertrophy. Finally, the role of Act as a regulator of muscle mass was supported by the observation that ActA overexpression induced muscle weight loss (-15%). In conclusion, our results show that satellite cell proliferation and both Mstn and Act inhibition are involved in the FS-induced muscle hypertrophy.

Simulation of Ground-Water Flow and Effects of Ground-Water Irrigation on Base Flow in the Elkhorn and Loup River Basins, Nebraska

USGS Publications Warehouse

Peterson, Steven M.; Stanton, Jennifer S.; Saunders, Amanda T.; Bradley, Jesse R.

2008-01-01

Irrigated agriculture is vital to the livelihood of communities in the Elkhorn and Loup River Basins in Nebraska, and ground water is used to irrigate most of the cropland. Concerns about the sustainability of ground-water and surface-water resources have prompted State and regional agencies to evaluate the cumulative effects of ground-water irrigation in this area. To facilitate understanding of the effects of ground-water irrigation, a numerical computer model was developed to simulate ground-water flow and assess the effects of ground-water irrigation (including ground-water withdrawals, hereinafter referred to as pumpage, and enhanced recharge) on stream base flow. The study area covers approximately 30,800 square miles, and includes the Elkhorn River Basin upstream from Norfolk, Nebraska, and the Loup River Basin upstream from Columbus, Nebraska. The water-table aquifer consists of Quaternary-age sands and gravels and Tertiary-age silts, sands, and gravels. The simulation was constructed using one layer with 2-mile by 2-mile cell size. Simulations were constructed to represent the ground-water system before 1940 and from 1940 through 2005, and to simulate hypothetical conditions from 2006 through 2045 or 2055. The first simulation represents steady-state conditions of the system before anthropogenic effects, and then simulates the effects of early surface-water development activities and recharge of water leaking from canals during 1895 to 1940. The first simulation ends at 1940 because before that time, very little pumpage for irrigation occurred, but after that time it became increasingly commonplace. The pre-1940 simulation was calibrated against measured water levels and estimated long-term base flow, and the 1940 through 2005 simulation was calibrated against measured water-level changes and estimated long-term base flow. The calibrated 1940 through 2005 simulation was used as the basis for analyzing hypothetical scenarios to evaluate the effects of ground-water irrigation on stream base flow for 1940 through 2005 and for 2006 through 2045. Simulated base flows were compared for scenarios that alternately did or did not include a representation of the effects of ground-water irrigation. The difference between simulated base flows for the two scenarios represents the predicted effects of ground-water irrigation on base flow. Comparison of base flows between simulations with ground-water irrigation and no ground-water irrigation indicated that ground-water irrigation has cumulatively reduced streamflows from 1940 through 2005 by 888,000 acre-feet in the Elkhorn River Basin and by 2,273,000 acre-feet in the Loup River Basin. Generally, predicted cumulative effects of ground-water irrigation on base flow were 5 to 10 times larger from 2006 through 2045 than from 1940 through 2005, and were 7,678,000 acre-feet for the Elkhorn River Basin and 14,784,000 acre-feet for the Loup River Basin. The calibrated simulation also was used to estimate base-flow depletion as a percentage of pumping volumes for a 50-year future time period, because base-flow depletion percentages are used to guide the placement of management boundaries in Nebraska. Mapped results of the base-flow depletion analysis conducted for most of the interior of the study area indicated that pumpage of one additional theoretical well simulated for a future 50-year period generally would result in more than 80 percent depletion when it was located close to the stream, except in areas where depletion was partly offset by reduced ground-water discharge to evapotranspiration in wetland areas. In many areas, depletion for the 50-year future period composed greater than 10 percent of the pumped water volume for theoretical wells placed less than 7 or 8 miles from the stream, though considerable variations existed because of the heterogeneity of the natural system represented in the simulation. For a few streams, predicted future simulated base flows dec

Carbon Nanotube Arrays for Intracellular Delivery and Biological Applications

NASA Astrophysics Data System (ADS)

Golshadi, Masoud

Introducing nucleic acids into mammalian cells is a crucial step to elucidate biochemical pathways, modify gene expression in immortalized cells, primary cells, and stem cells, and intoduces new approaches for clinical diagnostics and therapeutics. Current gene transfer technologies, including lipofection, electroporation, and viral delivery, have enabled break-through advances in basic and translational science to enable derivation and programming of embryonic stem cells, advanced gene editing using CRISPR (Clustered regularly interspaced short palindromic repeats), and development of targeted anti-tumor therapy using chimeric antigen receptors in T-cells (CAR-T). Despite these successes, current transfection technologies are time consuming and limited by the inefficient introduction of test molecules into large populations of target cells, and the cytotoxicity of the techniques. Moreover, many cell types cannot be consistently transfected by lipofection or electroporation (stem cells, T-cells) and viral delivery has limitations to the size of experimental DNA that can be packaged. In this dissertation, a novel coverslip-like platform consisting of an array of aligned hollow carbon nanotubes (CNTs) embedded in a sacrificial template is developed that enhances gene transfer capabilities, including high efficiency, low toxicity, in an expanded range of target cells, with the potential to transfer mixed combinations of protein and nucleic acids. The CNT array devices are fabricated by a scalable template-based manufacturing method using commercially available membranes, eliminating the need for nano-assembly. High efficient transfection has been demonstrated by delivering various cargos (nanoparticles, dye and plasmid DNA) into populations of cells, achieving 85% efficiency of plasmid DNA delivery into immortalized cells. Moreover, the CNT-mediated transfection of stem cells shows 3 times higher efficiency compared to current lipofection methods. Evaluating the cell-CNT interaction elucidates the importance of the geometrical properties of CNT arrays (CNT exposed length and surface morphology) on transfection efficiency. The results indicate that densely-packed and shortly-exposed CNT arrays with planar surface will enhance gene delivery using this new platform. This technology offers a significant increase in efficiency and cell viability, along with the ease of use compared to current standard methods, which demonstrates its potential to accelerate the development of new cell models to study intractable diseases, decoding the signaling pathways, and drug discovery.

Infection Risk From Conducted Electrical Weapon Probes: What Do We Know?

PubMed

Kroll, Mark W; Ritter, Mollie B; Guilbault, Richard A; Panescu, Dorin

2016-11-01

Concern has been raised over the infection risk of the TASER electrical weapon since the probes penetrate the skin. The manufacturing process produces unsterilized probes with a 5% rate of Staphylococcus aureus contamination. Voluntary recipients (n = 208) of probe exposures were surveyed and there were no self-observations of infection. With over 3.3 million probe landings, there have been 10 case reports of penetrations of sensitive tissue with no reported infections. The electrical field was modeled and found that the electrical pulses generate a field of over 1200 V/mm on the dart portion. This is sufficient to sterilize the dart via electroporation. Electrical weapon probes appear to have a very low (possibly zero) rate of infection. The factors leading to this low infection rate appear to be a manufacturing process producing a low rate of bacterial contamination and the pulses sterilizing the dart via electroporation. © 2016 American Academy of Forensic Sciences.

Analysis of Alternative Pre-RNA Splicing in the Mouse Retina Using a Fluorescent Reporter.

PubMed

Murphy, Daniel; Kolandaivelu, Saravanan; Ramamurthy, Visvanathan; Stoilov, Peter

2016-01-01

In vivo alternative splicing is controlled in a tissue and cell type specific manner. Often individual cellular components of complex tissues will express different splicing programs. Thus, when studying splicing in multicellular organisms it is critical to determine the exon inclusion levels in individual cells positioned in the context of their native tissue or organ. Here we describe how a fluorescent splicing reporter in combination with in vivo electroporation can be used to visualize alternative splicing in individual cells within mature tissues. In a test case we show how the splicing of a photoreceptor specific exon can be visualized within the mouse retina. The retina was chosen as an example of a complex tissue that is fragile and whose cells cannot be studied in culture. With minor modifications to the injection and electroporation procedure, the protocol we outline can be applied to other tissues and organs.

Percutaneous Irreversible Electroporation of a Large Centrally Located Hepatocellular Adenoma in a Woman with a Pregnancy Wish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scheffer, Hester J., E-mail: hj.scheffer@vumc.nl; Melenhorst, Marleen C. A. M., E-mail: m.melenhorst@vumc.nl; Tilborg, Aukje A. J. M. van, E-mail: a.vantilborg@vumc.nl

Irreversible electroporation (IRE) is a novel image-guided ablation technique that is rapidly gaining popularity in the treatment of malignant liver tumors located near large vessels or bile ducts. We describe a 28-year-old female patient with a 5 cm large, centrally located hepatocellular adenoma who wished to get pregnant. Regarding the risk of growth and rupture of the adenoma caused by hormonal changes during pregnancy, treatment of the tumor was advised prior to pregnancy. However, due to its central location, the tumor was considered unsuitable for resection and thermal ablation. Percutaneous CT-guided IRE was performed without complications and led to rapid andmore » impressive tumor shrinkage. Subsequent pregnancy and delivery went uncomplicated. This case report suggests that the indication for IRE may extend to the treatment of benign liver tumors that cannot be treated safely otherwise.« less

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

PubMed Central

Molotkov, Dmitry A.; Yukin, Alexey Y.; Afzalov, Ramil A.; Khiroug, Leonard S.

2010-01-01

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells. The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices. PMID:20972387

Polymer multilayer tattooing for enhanced DNA vaccination

NASA Astrophysics Data System (ADS)

Demuth, Peter C.; Min, Younjin; Huang, Bonnie; Kramer, Joshua A.; Miller, Andrew D.; Barouch, Dan H.; Hammond, Paula T.; Irvine, Darrell J.

2013-04-01

DNA vaccines have many potential benefits but have failed to generate robust immune responses in humans. Recently, methods such as in vivo electroporation have demonstrated improved performance, but an optimal strategy for safe, reproducible, and pain-free DNA vaccination remains elusive. Here we report an approach for rapid implantation of vaccine-loaded polymer films carrying DNA, immune-stimulatory RNA, and biodegradable polycations into the immune-cell-rich epidermis, using microneedles coated with releasable polyelectrolyte multilayers. Films transferred into the skin following brief microneedle application promoted local transfection and controlled the persistence of DNA and adjuvants in the skin from days to weeks, with kinetics determined by the film composition. These ‘multilayer tattoo’ DNA vaccines induced immune responses against a model HIV antigen comparable to electroporation in mice, enhanced memory T-cell generation, and elicited 140-fold higher gene expression in non-human primate skin than intradermal DNA injection, indicating the potential of this strategy for enhancing DNA vaccination.

Computed Flow Through An Artificial Heart Valve

NASA Technical Reports Server (NTRS)

Rogers, Stewart E.; Kwak, Dochan; Kiris, Cetin; Chang, I-Dee

1994-01-01

Report discusses computations of blood flow through prosthetic tilting disk valve. Computational procedure developed in simulation used to design better artificial hearts and valves by reducing or eliminating following adverse flow characteristics: large pressure losses, which prevent hearts from working efficiently; separated and secondary flows, which causes clotting; and high turbulent shear stresses, which damages red blood cells. Report reiterates and expands upon part of NASA technical memorandum "Computed Flow Through an Artificial Heart and Valve" (ARC-12983). Also based partly on research described in "Numerical Simulation of Flow Through an Artificial Heart" (ARC-12478).

Base-flow data in the Arnold Air Force Base area, Tennessee, June and October 2002

USGS Publications Warehouse

Robinson, John A.; Haugh, Connor J.

2004-01-01

Arnold Air Force Base (AAFB) occupies about 40,000 acres in Coffee and Franklin Counties, Tennessee. The primary mission of AAFB is to support the development of aerospace systems. This mission is accomplished through test facilities at Arnold Engineering Development Center (AEDC), which occupies about 4,000 acres in the center of AAFB. Base-flow data including discharge, temperature, and specific conductance were collected for basins in and near AAFB during high base-flow and low base-flow conditions. Data representing high base-flow conditions from 109 sites were collected on June 3 through 5, 2002, when discharge measurements at sites with flow ranged from 0.005 to 46.4 ft3/s. Data representing low base-flow conditions from 109 sites were collected on October 22 and 23, 2002, when discharge measurements at sites with flow ranged from 0.02 to 44.6 ft3/s. Discharge from the basin was greater during high base-flow conditions than during low base-flow conditions. In general, major tributaries on the north side and southeastern side of the study area (Duck River and Bradley Creek, respectively) had the highest flows during the study. Discharge data were used to categorize stream reaches and sub-basins. Stream reaches were categorized as gaining, losing, wet, dry, or unobserved for each base-flow measurement period. Gaining stream reaches were more common during the high base-flow period than during the low base-flow period. Dry stream reaches were more common during the low base-flow period than during the high base-flow period. Losing reaches were more predominant in Bradley Creek and Crumpton Creek. Values of flow per square mile for the study area of 0.55 and 0.37 (ft3/s)/mi2 were calculated using discharge data collected on June 3 through 5, 2002, and October 22 and 23, 2002, respectively. Sub-basin areas with surplus or deficient flow were defined within the basin. Drainage areas for each stream measurement site were delineated and measured from topographic maps. Change in flow per square mile for each sub-basin was calculated using data from each base-flow measurement period. The calculated values were used to define the areas of surplus or deficient flow for high and low base-flow conditions. Many areas of deficient flow were present throughout the study area under high and low base-flow conditions. Most areas of deficient flow were in the headwater basins. Fewer areas of surplus flow were present under low base-flow conditions than during the high base-flow conditions. The flow per square mile for each major tributary basin in the study area also was calculated. The values of flow per square mile for the Dry Creek, Spring Creek, and Wiley Creek basins were greatest under both high and low base-flow conditions.

Fluid-cooled heat sink with improved fin areas and efficiencies for use in cooling various devices

DOEpatents

Bharathan, Desikan; Bennion, Kevin; Kelly, Kenneth; Narumanchi, Sreekant

2015-04-21

The disclosure provides a fluid-cooled heat sink having a heat transfer base and a plurality of heat transfer fins in thermal communication with the heat transfer base, where the heat transfer base and the heat transfer fins form a central fluid channel through which a forced or free cooling fluid may flow. The heat transfer pins are arranged around the central fluid channel with a flow space provided between adjacent pins, allowing for some portion of the central fluid channel flow to divert through the flow space. The arrangement reduces the pressure drop of the flow through the fins, optimizes average heat transfer coefficients, reduces contact and fin-pin resistances, and reduces the physical footprint of the heat sink in an operating environment.

Electroporation followed by electrochemical measurement of quantal transmitter release from single cells using a patterned microelectrode.

PubMed

Ghosh, Jaya; Liu, Xin; Gillis, Kevin D

2013-06-07

An electrochemical microelectrode located immediately adjacent to a single neuroendocrine cell can record spikes of amperometric current that result from exocytosis of oxidizable transmitter from individual vesicles, i.e., quantal exocytosis. Here, we report the development of an efficient method where the same electrochemical microelectrode is used to electropermeabilize an adjacent chromaffin cell and then measure the consequent quantal catecholamine release using amperometry. Trains of voltage pulses, 5-7 V in amplitude and 0.1-0.2 ms in duration, were used to reliably trigger release from cells using gold electrodes. Amperometric spikes induced by electropermeabilization had similar areas, peak heights and durations as amperometric spikes elicited by depolarizing high K(+) solutions, therefore release occurs from individual secretory granules. Uptake of trypan blue stain into cells demonstrated that the plasma membrane is permeabilized by the voltage stimulus. Voltage pulses did not degrade the electrochemical sensitivity of the electrodes assayed using a test analyte. Surprisingly, robust quantal release was elicited upon electroporation in the absence of Ca(2+) in the bath solution (0 Ca(2+)/5 mM EGTA). In contrast, electropermeabilization-induced transmitter release required Cl(-) in the bath solution in that bracketed experiments demonstrated a steep dependence of the rate of electropermeabilization-induced transmitter release on [Cl(-)] between 2 and 32 mM. Using the same electrochemical electrode to electroporate and record quantal release of catecholamines from an individual chromaffin cell allows precise timing of the stimulus, stimulation of a single cell at a time, and can be used to load membrane-impermeant substances into a cell.

An Engineered Membrane to Measure Electroporation: Effect of Tethers and Bioelectronic Interface

PubMed Central

Hoiles, William; Krishnamurthy, Vikram; Cranfield, Charles G.; Cornell, Bruce

2014-01-01

This article reports on the construction and predictive models for a platform comprised of an engineered tethered membrane. The platform provides a controllable and physiologically relevant environment for the study of the electroporation process. The mixed self-assembled membrane is formed via a rapid solvent exchange technique. The membrane is tethered to the gold electrode and includes an ionic reservoir separating the membrane and gold surface. Above the membrane, there is an electrolyte solution, and a gold counterelectrode. A voltage is applied between the gold electrodes and the current measured. The current is dependent on the energy required to form aqueous pores and the conductance of each pore. A two-level predictive model, consisting of a macroscopic and a continuum model, is developed to relate the pore dynamics to the measured current. The macroscopic model consists of an equivalent circuit model of the tethered membrane, and asymptotic approximations to the Smoluchowski-Einstein equation of electroporation that is dependent on the pore conductance and the energy required to form aqueous pores. The continuum model is a generalized Poisson-Nernst-Planck (GPNP) system where an activity coefficient to account for steric effects of ions is added to the standard PNP system. The GPNP is used to evaluate the conductance of aqueous pores, and the electrical energy required to form the pores. As an outcome of the setup of the device and the two-level model, biologically important variables can be estimated from experimental measurements. To validate the accuracy of the two-level model, the predicted current is compared with experimentally measured current for different tethering densities. PMID:25229142

The second phase of bipolar, nanosecond-range electric pulses determines the electroporation efficiency.

PubMed

Pakhomov, Andrei G; Grigoryev, Sergey; Semenov, Iurii; Casciola, Maura; Jiang, Chunqi; Xiao, Shu

2018-03-29

Bipolar cancellation refers to a phenomenon when applying a second electric pulse reduces ("cancels") cell membrane damage by a preceding electric pulse of the opposite polarity. Bipolar cancellation is a reason why bipolar nanosecond electric pulses (nsEP) cause weaker electroporation than just a single unipolar phase of the same pulse. This study was undertaken to explore the dependence of bipolar cancellation on nsEP parameters, with emphasis on the amplitude ratio of two opposite polarity phases of a bipolar pulse. Individual cells (CHO, U937, or adult mouse ventricular cardiomyocytes (VCM)) were exposed to either uni- or bipolar trapezoidal nsEP, or to nanosecond electric field oscillations (NEFO). The membrane injury was evaluated by time-lapse confocal imaging of the uptake of propidium (Pr) or YO-PRO-1 (YP) dyes and by phosphatidylserine (PS) externalization. Within studied limits, bipolar cancellation showed little or no dependence on the electric field intensity, pulse repetition rate, chosen endpoint, or cell type. However, cancellation could increase for larger pulse numbers and/or for longer pulses. The sole most critical parameter which determines bipolar cancellation was the phase ratio: maximum cancellation was observed with the 2nd phase of about 50% of the first one, whereas a larger 2nd phase could add a damaging effect of its own. "Swapping" the two phases, i.e., delivering the smaller phase before the larger one, reduced or eliminated cancellation. These findings are discussed in the context of hypothetical mechanisms of bipolar cancellation and electroporation by nsEP. Copyright © 2018 Elsevier B.V. All rights reserved.

HIV-DNA Given with or without Intradermal Electroporation Is Safe and Highly Immunogenic in Healthy Swedish HIV-1 DNA/MVA Vaccinees: A Phase I Randomized Trial.

PubMed

Nilsson, Charlotta; Hejdeman, Bo; Godoy-Ramirez, Karina; Tecleab, Teghesti; Scarlatti, Gabriella; Bråve, Andreas; Earl, Patricia L; Stout, Richard R; Robb, Merlin L; Shattock, Robin J; Biberfeld, Gunnel; Sandström, Eric; Wahren, Britta

2015-01-01

We compared safety and immunogenicity of intradermal (ID) vaccination with and without electroporation (EP) in a phase I randomized placebo-controlled trial of an HIV-DNA prime HIV-MVA boost vaccine in healthy Swedish volunteers. HIV-DNA plasmids encoding HIV-1 genes gp160 subtypes A, B and C; Rev B; Gag A and B and RTmut B were given ID at weeks 0, 6 and 12 in a dose of 0.6 mg. Twenty-five volunteers received vaccine using a needle-free device (ZetaJet) with (n=16) or without (n=9) ID EP (Dermavax). Five volunteers were placebo recipients. Boosting with recombinant MVA-CMDR expressing HIV-1 Env, Gag, Pol of CRF01_AE (HIV-MVA) or placebo was performed at weeks 24 and 40. Nine of the vaccinees received a subtype C CN54 gp140 protein boost together with HIV-MVA. The ID/EP delivery was very well tolerated. After three HIV-DNA immunizations, no statistically significant difference was seen in the IFN-γ ELISpot response rate to Gag between HIV-DNA ID/EP recipients (5/15, 33%) and HIV-DNA ID recipients (1/7, 14%, p=0.6158). The first HIV-MVA or HIV-MVA+gp140 vaccination increased the IFN-γ ELISpot response rate to 18/19 (95%). CD4+ and/or CD8+ T cell responses to Gag or Env were demonstrable in 94% of vaccinees. A balanced CD4+ and CD8+ T cell response was noted, with 78% and 71% responders, respectively. IFN-γ and IL-2 dominated the CD4+ T cell response to Gag and Env. The CD8+ response to Gag was broader with expression of IFN-γ, IL-2, MIP-1β and/or CD107. No differences were seen between DNA vaccine groups. Binding antibodies were induced after the second HIV-MVA+/-gp140 in 93% of vaccinees to subtype C Env, with the highest titers among EP/gp140 recipients. Intradermal electroporation of HIV-DNA was well tolerated. Strong cell- and antibody-mediated immune responses were elicited by the HIV-DNA prime and HIV-MVA boosting regimen, with or without intradermal electroporation use. International Standard Randomised Controlled Trial Number (ISRCTN) 60284968.

HIV-DNA Given with or without Intradermal Electroporation Is Safe and Highly Immunogenic in Healthy Swedish HIV-1 DNA/MVA Vaccinees: A Phase I Randomized Trial

PubMed Central

Nilsson, Charlotta; Hejdeman, Bo; Godoy-Ramirez, Karina; Tecleab, Teghesti; Scarlatti, Gabriella; Bråve, Andreas; Earl, Patricia L.; Stout, Richard R.; Robb, Merlin L.; Shattock, Robin J.; Biberfeld, Gunnel; Sandström, Eric; Wahren, Britta

2015-01-01

Background We compared safety and immunogenicity of intradermal (ID) vaccination with and without electroporation (EP) in a phase I randomized placebo-controlled trial of an HIV-DNA prime HIV-MVA boost vaccine in healthy Swedish volunteers. Methods HIV-DNA plasmids encoding HIV-1 genes gp160 subtypes A, B and C; Rev B; Gag A and B and RTmut B were given ID at weeks 0, 6 and 12 in a dose of 0.6 mg. Twenty-five volunteers received vaccine using a needle-free device (ZetaJet) with (n=16) or without (n=9) ID EP (Dermavax). Five volunteers were placebo recipients. Boosting with recombinant MVA-CMDR expressing HIV-1 Env, Gag, Pol of CRF01_AE (HIV-MVA) or placebo was performed at weeks 24 and 40. Nine of the vaccinees received a subtype C CN54 gp140 protein boost together with HIV-MVA. Results The ID/EP delivery was very well tolerated. After three HIV-DNA immunizations, no statistically significant difference was seen in the IFN-γ ELISpot response rate to Gag between HIV-DNA ID/EP recipients (5/15, 33%) and HIV-DNA ID recipients (1/7, 14%, p=0.6158). The first HIV-MVA or HIV-MVA+gp140 vaccination increased the IFN-γ ELISpot response rate to 18/19 (95%). CD4+ and/or CD8+ T cell responses to Gag or Env were demonstrable in 94% of vaccinees. A balanced CD4+ and CD8+ T cell response was noted, with 78% and 71% responders, respectively. IFN-γ and IL-2 dominated the CD4+ T cell response to Gag and Env. The CD8+ response to Gag was broader with expression of IFN-γ, IL-2, MIP-1β and/or CD107. No differences were seen between DNA vaccine groups. Binding antibodies were induced after the second HIV-MVA+/-gp140 in 93% of vaccinees to subtype C Env, with the highest titers among EP/gp140 recipients. Conclusion Intradermal electroporation of HIV-DNA was well tolerated. Strong cell- and antibody-mediated immune responses were elicited by the HIV-DNA prime and HIV-MVA boosting regimen, with or without intradermal electroporation use. Trial Registration International Standard Randomised Controlled Trial Number (ISRCTN) 60284968 PMID:26121679

Observations of Gas-Liquid Flows Through Contractions in Microgravity

NASA Technical Reports Server (NTRS)

McQuillen, John

1996-01-01

Tests were conducted for an air-water flow through two sudden contractions aboard the NASA DC-9 low gravity aircraft. Flow rate, residual accelerations, void fraction, film thickness, and pressure drop data were recorded and flow visualization at 250 images per second were recorded. Some preliminary results based on the flow visualization data are presented for bubbly, slug and annular flow.

Precision cancer immunotherapy: optimizing dendritic cell-based strategies to induce tumor antigen-specific T-cell responses against individual patient tumors.

PubMed

Osada, Takuya; Nagaoka, Koji; Takahara, Masashi; Yang, Xiao Yi; Liu, Cong-Xiao; Guo, Hongtao; Roy Choudhury, Kingshuk; Hobeika, Amy; Hartman, Zachary; Morse, Michael A; Lyerly, H Kim

2015-05-01

Most dendritic cell (DC)-based vaccines have loaded the DC with defined antigens, but loading with autologos tumor-derived antigens would generate DCs that activate personalized tumor-specific T-cell responses. We hypothesized that DC matured with an optimized combination of reagents and loaded with tumor-derived antigens using a clinically feasible electroporation strategy would induce potent antitumor immunity. We first studied the effects on DC maturation and antigen presentation of the addition of picibanil (OK432) to a combination of zoledronic acid, tumor necrosis factor-α, and prostaglandin E2. Using DC matured with the optimized combination, we tested 2 clinically feasible sources of autologous antigen for electroloading, total tumor mRNA or total tumor lysate, to determine which stimulated more potent antigen-specific T cells in vitro and activated more potent antitumor immunity in vivo. The combination of tumor necrosis factor-α/prostaglandin E2/zoledronic acid/OK432 generated DC with high expression of maturation markers and antigen-specific T-cell stimulatory function in vitro. Mature DC electroloaded with tumor-derived mRNA [mRNA electroporated dendritic cell (EPDC)] induced greater expansion of antigen-specific T cells in vitro than DC electroloaded with tumor lysate (lysate EPDC). In a therapeutic model of MC38-carcinoembryonic antigen colon cancer-bearing mice, vaccination with mRNA EPDC induced the most efficient anti-carcinoembryonic antigen cellular immune response, which significantly suppressed tumor growth. In conclusion, mature DC electroloaded with tumor-derived mRNA are a potent cancer vaccine, especially useful when specific tumor antigens for vaccination have not been identified, allowing autologous tumor, and if unavailable, allogeneic cell lines to be used as an unbiased source of antigen. Our data support clinical testing of this strategy.

Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers.

PubMed

Gallego-Perez, Daniel; Otero, Jose J; Czeisler, Catherine; Ma, Junyu; Ortiz, Cristina; Gygli, Patrick; Catacutan, Fay Patsy; Gokozan, Hamza Numan; Cowgill, Aaron; Sherwood, Thomas; Ghatak, Subhadip; Malkoc, Veysi; Zhao, Xi; Liao, Wei-Ching; Gnyawali, Surya; Wang, Xinmei; Adler, Andrew F; Leong, Kam; Wulff, Brian; Wilgus, Traci A; Askwith, Candice; Khanna, Savita; Rink, Cameron; Sen, Chandan K; Lee, L James

2016-02-01

Safety concerns and/or the stochastic nature of current transduction approaches have hampered nuclear reprogramming's clinical translation. We report a novel non-viral nanotechnology-based platform permitting deterministic large-scale transfection with single-cell resolution. The superior capabilities of our technology are demonstrated by modification of the well-established direct neuronal reprogramming paradigm using overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM). Reprogramming efficiencies were comparable to viral methodologies (up to ~9-12%) without the constraints of capsid size and with the ability to control plasmid dosage, in addition to showing superior performance relative to existing non-viral methods. Furthermore, increased neuronal complexity could be tailored by varying BAM ratio and by including additional proneural genes to the BAM cocktail. Furthermore, high-throughput NEP allowed easy interrogation of the reprogramming process. We discovered that BAM-mediated reprogramming is regulated by AsclI dosage, the S-phase cyclin CCNA2, and that some induced neurons passed through a nestin-positive cell stage. In the field of regenerative medicine, the ability to direct cell fate by nuclear reprogramming is an important facet in terms of clinical application. In this article, the authors described their novel technique of cell reprogramming through overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM) by in situ electroporation through nanochannels. This new technique could provide a platform for further future designs. Copyright © 2016 Elsevier Inc. All rights reserved.

Fluid-cooled heat sink for use in cooling various devices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bharathan, Desikan; Bennion, Kevin; Kelly, Kenneth

The disclosure provides a fluid-cooled heat sink having a heat transfer base, a shroud, and a plurality of heat transfer fins in thermal communication with the heat transfer base and the shroud, where the heat transfer base, heat transfer fins, and the shroud form a central fluid channel through which a forced or free cooling fluid may flow. The heat transfer pins are arranged around the central fluid channel with a flow space provided between adjacent pins, allowing for some portion of the central fluid channel flow to divert through the flow space. The arrangement reduces the pressure drop ofmore » the flow through the fins, optimizes average heat transfer coefficients, reduces contact and fin-pin resistances, and reduces the physical footprint of the heat sink in an operating environment.« less

New strategies for genetic engineering Pseudomonas syringae using recombination

USDA-ARS?s Scientific Manuscript database

Here we report that DNA oligonucleotides (oligos) introduced directly into bacteria by electroporation can recombine with the bacterial chromosome. This phenomenon was identified in Pseudomonas syringae and we subsequently found that Escherichia coli, Salmonella typhimurium and Shigella flexneri are...

Hydrogeologic investigations of the Sierra Vista subwatershed of the Upper San Pedro Basin, Cochise County, southeast Arizona

USGS Publications Warehouse

Pool, Donald R.; Coes, Alissa L.

1999-01-01

The hydrogeologic system in the Sierra Vista subwatershed of the Upper San Pedro Basin in southeastern Arizona was investigated for the purpose of developing a better understanding of stream-aquifer interactions. The San Pedro River is an intermittent stream that supports a narrow corridor of riparian vegetation. Withdrawal of ground water will result in reduced discharge from the basin through reduced base flow and evapotranspiration; however, the rate and location of reduced discharge are uncertain. The investigation resulted in better definition of distributions of silt and clay in the regional aquifer; changes in seasonal precipitation, runoff, and base flow in the San Pedro River; sources of base flow; and regional water-level changes. Regional ground-water flow is separated into deep-confined and shallow-unconfined systems by silt and clay. Precipitation, runoff, and base flow declined at the Charleston streamflow-gaging station from 1936 through 1997 for the months of June through October. Base flow at the Charleston station during 1996 and 1997 was primarily supplied by ground water recharged near the San Pedro River during recent major runoff and by minor contributions from the regional aquifer. The decline in base flow, about 2 cubic feet per second, has several probable causes including declining runoff and recharge near the river during June through October and increased interception of ground-water flow to the river by wells and phreatophytes. Water levels in wells throughout the regional aquifer generally declined at rates of 0.2 to 0.5 feet per year between 1940 and the mid-1980's, which corresponded with a period of below-average winter precipitation. Water levels in wells in the Fort Huachuca and Sierra Vista areas declined at rates that were faster than regional rates of decline through 1998 and caused diversion of ground-water flow that would have discharged along perennial stream reaches.

Functional evaluation of malaria Pfs25 DNA vaccine by in vivo electroporation in olive baboons.

PubMed

Kumar, Rajesh; Nyakundi, Ruth; Kariuki, Thomas; Ozwara, Hastings; Nyamongo, Onkoba; Mlambo, Godfree; Ellefsen, Barry; Hannaman, Drew; Kumar, Nirbhay

2013-06-28

Plasmodium falciparum Pfs25 antigen, expressed on the surface of zygotes and ookinetes, is one of the leading targets for the development of a malaria transmission-blocking vaccine (TBV). Our laboratory has been evaluating DNA plasmid based Pfs25 vaccine in mice and non-human primates. Previously, we established that in vivo electroporation (EP) delivery is an effective method to improve the immunogenicity of DNA vaccine encoding Pfs25 in mice. In order to optimize the in vivo EP procedure and test for its efficacy in more clinically relevant larger animal models, we employed in vivo EP to evaluate the immune response and protective efficacy of Pfs25 encoding DNA vaccine in nonhuman primates (olive baboons, Papio anubis). The results showed that at a dose of 2.5mg DNA vaccine, antibody responses were significantly enhanced with EP as compared to without EP resulting in effective transmission blocking efficiency. Similar immunogenicity enhancing effect of EP was also observed with lower doses (0.5mg and 1mg) of DNA plasmids. Further, final boosting with a single dose of recombinant Pfs25 protein resulted in dramatically enhanced antibody titers and significantly increased functional transmission blocking efficiency. Our study suggests priming with DNA vaccine via EP along with protein boost regimen as an effective method to elicit potent immunogenicity of malaria DNA vaccines in nonhuman primates and provides the basis for further evaluation in human volunteers. Copyright © 2013 Elsevier Ltd. All rights reserved.

Combination of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 technique with the piggybac transposon system for mouse in utero electroporation to study cortical development.

PubMed

Cheng, Man; Jin, Xubin; Mu, Lili; Wang, Fangyu; Li, Wei; Zhong, Xiaoling; Liu, Xuan; Shen, Wenchen; Liu, Ying; Zhou, Yan

2016-09-01

In utero electroporation (IUE) is commonly used to study cortical development of cerebrum by downregulating or overexpressing genes of interest in neural progenitor cells (NPCs) of small mammals. However, exogenous plasmids are lost or diluted over time. Furthermore, gene knockdown based on short-hairpin RNAs may exert nonspecific effects that lead to aberrant neuronal migration. Genomic engineering by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has great research and therapeutic potentials. Here we integrate the CRISPR/Cas9 components into the piggyBac (PB) transposon system (the CRISPR/Cas9-PB toolkit) for cortical IUEs. The mouse Sry-related HMG box-2 (Sox2) gene was selected as the target for its application. Most transduced cortical NPCs were depleted of SOX2 protein as early as 3 days post-IUE, whereas expressions of SOX1 and PAX6 remained intact. Furthermore, both the WT Cas9 and the D10A nickase mutant Cas9n showed comparable knockout efficiency. Transduced cortical cells were purified with fluorescence-activated cell sorting, and effective gene editing at the Sox2 loci was confirmed. Thus, application of the CRISPR/Cas9-PB toolkit in IUE is a promising strategy to study gene functions in cortical NPCs and their progeny. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Parametric study of irreversible electroporation with different needle electrodes: electrical and thermal analysis.

PubMed

Nickfarjam, Abolfazl; Firoozabadi, S Mohammad P

2014-08-01

Irreversible electroporation (IRE) is a new tumour ablation method used in cancer treatment procedures. In a successful IRE treatment it is crucial to impose minimum thermal damage to the tumour and its surrounding healthy tissue, while subjecting the entire tumour to a strong electric field. Here we present a 3D model of a subcutaneous tumour in a four-layer skin using a geometry-based finite element approach. Four common needle electrode configurations were studied in this paper. The study evaluated six essential factors which are important in the electrical and thermal distributions in tumour and normal tissue. The results revealed that a hexagonal 3 × 3 geometry provides the maximum electrical coverage of the tumour, compared to other electrode configurations. However, in some cases the hexagonal 2 × 2 geometry can ablate the entire tumour with less damage to normal tissue. We found that the deeper insertion of 2- and 4-electrode geometries can lead to more damage to healthy tissue. The results also indicate that the insertion of the electrodes into tumour tissue can increase thermal damage dramatically due to existing large electrical conductivity. These findings suggest that needle electrodes should not be placed within the tumour tissue if the goal is to prevent thermal damage. This method can be used as a trade-off between electric field coverage in tumour tissue and thermal damage to both tumour and normal tissue.

Nonintrusive Flow Rate Determination Through Space Shuttle Water Coolant Loop Floodlight Coldplate

NASA Technical Reports Server (NTRS)

Werlink, Rudolph; Johnson, Harry; Margasahayam, Ravi

1997-01-01

Using a Nonintrusive Flow Measurement System (NFMS), the flow rates through the Space Shuttle water coolant coldplate were determined. The objective of this in situ flow measurement was to prove or disprove a potential block inside the affected coldplate had contributed to a reduced flow rate and the subsequent ice formation on the Space Shuttle Discovery. Flow through the coldplate was originally calculated to be 35 to 38 pounds per hour. This application of ultrasonic technology advanced the envelope of flow measurements through use of 1/4-inch-diameter tubing, which resulted in extremely low flow velocities (5 to 30 pounds per hour). In situ measurements on the orbiters Discovery and Atlantis indicated both vehicles, on the average, experienced similar flow rates through the coldplate (around 25 pounds per hour), but lower rates than the designed flow. Based on the noninvasive checks, further invasive troubleshooting was eliminated. Permanent monitoring using the NFMS was recommended.

Effect of ac electric field on the dynamics of a vesicle under shear flow in the small deformation regime

NASA Astrophysics Data System (ADS)

Sinha, Kumari Priti; Thaokar, Rochish M.

2018-03-01

Vesicles or biological cells under simultaneous shear and electric field can be encountered in dielectrophoretic devices or designs used for continuous flow electrofusion or electroporation. In this work, the dynamics of a vesicle subjected to simultaneous shear and uniform alternating current (ac) electric field is investigated in the small deformation limit. The coupled equations for vesicle orientation and shape evolution are derived theoretically, and the resulting nonlinear equations are handled numerically to generate relevant phase diagrams that demonstrate the effect of electrical parameters on the different dynamical regimes such as tank treading (TT), vacillating breathing (VB) [called trembling (TR) in this work], and tumbling (TU). It is found that while the electric Mason number (Mn), which represents the relative strength of the electrical forces to the shear forces, promotes the TT regime, the response itself is found to be sensitive to the applied frequency as well as the conductivity ratio. While higher outer conductivity promotes orientation along the flow axis, orientation along the electric field is favored when the inner conductivity is higher. Similarly a switch of orientation from the direction of the electric field to the direction of flow is possible by a mere change of frequency when the outer conductivity is higher. Interestingly, in some cases, a coupling between electric field-induced deformation and shear can result in the system admitting an intermediate TU regime while attaining the TT regime at high Mn. The results could enable designing better dielectrophoretic devices wherein the residence time as well as the dynamical states of the vesicular suspension can be controlled as per the application.

Time-Dependent Impact of Irreversible Electroporation on Pancreas, Liver, Blood Vessels and Nerves: A Systematic Review of Experimental Studies.

PubMed

Vogel, J A; van Veldhuisen, E; Agnass, P; Crezee, J; Dijk, F; Verheij, J; van Gulik, T M; Meijerink, M R; Vroomen, L G; van Lienden, K P; Besselink, M G

2016-01-01

Irreversible electroporation (IRE) is a novel ablation technique in the treatment of unresectable cancer. The non-thermal mechanism is thought to cause mostly apoptosis compared to necrosis in thermal techniques. Both in experimental and clinical studies, a waiting time between ablation and tissue or imaging analysis to allow for cell death through apoptosis, is often reported. However, the dynamics of the IRE effect over time remain unknown. Therefore, this study aims to summarize these effects in relation to the time between treatment and evaluation. A systematic search was performed in Pubmed, Embase and the Cochrane Library for original articles using IRE on pancreas, liver or surrounding structures in animal or human studies. Data on pathology and time between IRE and evaluation were extracted. Of 2602 screened studies, 36 could be included, regarding IRE in liver (n = 24), pancreas (n = 4), blood vessels (n = 4) and nerves (n = 4) in over 440 animals (pig, rat, goat and rabbit). No eligible human studies were found. In liver and pancreas, the first signs of apoptosis and haemorrhage were observed 1-2 hours after treatment, and remained visible until 24 hours in liver and 7 days in pancreas after which the damaged tissue was replaced by fibrosis. In solitary blood vessels, the tunica media, intima and lumen remained unchanged for 24 hours. After 7 days, inflammation, fibrosis and loss of smooth muscle cells were demonstrated, which persisted until 35 days. In nerves, the median time until demonstrable histological changes was 7 days. Tissue damage after IRE is a dynamic process with remarkable time differences between tissues in animals. Whereas pancreas and liver showed the first damages after 1-2 hours, this took 24 hours in blood vessels and 7 days in nerves.

From Signature-Based Towards Behaviour-Based Anomaly Detection (Extended Abstract)

DTIC Science & Technology

2010-11-01

data acquisition can serve as sensors. De- facto standard for IP flow monitoring is NetFlow format. Although NetFlow was originally developed by Cisco...packets with some common properties that pass through a network device. These collected flows are exported to an external device, the NetFlow ...Thanks to the network-based approach using NetFlow data, the detection algorithm is host independent and highly scalable. Deep Packet Inspection

An "Off-the-Shelf" System for Intraprocedural Electrical Current Evaluation and Monitoring of Irreversible Electroporation Therapy.

PubMed

Neal, Robert E; Kavnoudias, Helen; Thomson, Kenneth R

2015-06-01

Irreversible electroporation (IRE) ablation uses a series of brief electric pulses to create nanoscale defects in cell membranes, killing the cells. It has shown promise in numerous soft-tissue tumor applications. Larger voltages between electrodes will increase ablation volume, but exceeding electrical limits may risk damage to the patient, cause ineffective therapy delivery, or require generator restart. Monitoring electrical current for these conditions in real-time enables managing these risks. This capacity is not presently available in clinical IRE generators. We describe a system using a Tektronix TCP305 AC/DC Current Probe connected to a TCPA300 AC/DC Current Probe Amplifier, which is read on a computer using a Protek DSO-2090 USB computer-interfacing oscilloscope. Accuracy of the system was tested with a resistor circuit and by comparing measured currents with final outputs from the NanoKnife clinical electroporation pulse generator. Accuracy of measured currents was 1.64 ± 2.4 % relative to calculations for the resistor circuit and averaged 0.371 ± 0.977 % deviation from the NanoKnife. During clinical pulse delivery, the system offers real-time evaluation of IRE procedure progress and enables a number of methods for identifying approaching issues from electrical behavior of therapy delivery, facilitating protocol changes before encountering therapy delivery issues. This system can monitor electrical currents in real-time without altering the electric pulses or modifying the pulse generator. This facilitates delivering electric pulse protocols that remain within the optimal range of electrical currents-sufficient strength for clinically relevant ablation volumes, without the risk of exceeding safe electric currents or causing inadequate ablation.

Evaluation of Soft Tissue Sarcoma Tumors Electrical Conductivity Anisotropy Using Diffusion Tensor Imaging for Numerical Modeling on Electroporation.

PubMed

Ghazikhanlou-Sani, K; Firoozabadi, S M P; Agha-Ghazvini, L; Mahmoodzadeh, H

2016-06-01

There is many ways to assessing the electrical conductivity anisotropy of a tumor. Applying the values of tissue electrical conductivity anisotropy is crucial in numerical modeling of the electric and thermal field distribution in electroporation treatments. This study aims to calculate the tissues electrical conductivity anisotropy in patients with sarcoma tumors using diffusion tensor imaging technique. A total of 3 subjects were involved in this study. All of patients had clinically apparent sarcoma tumors at the extremities. The T1, T2 and DTI images were performed using a 3-Tesla multi-coil, multi-channel MRI system. The fractional anisotropy (FA) maps were performed using the FSL (FMRI software library) software regarding the DTI images. The 3D matrix of the FA maps of each area (tumor, normal soft tissue and bone/s) was reconstructed and the anisotropy matrix was calculated regarding to the FA values. The mean FA values in direction of main axis in sarcoma tumors were ranged between 0.475-0.690.  With assumption of isotropy of the electrical conductivity, the FA value of electrical conductivity at each X, Y and Z coordinate axes would be equal to 0.577. The gathered results showed that there is a mean error band of 20% in electrical conductivity, if the electrical conductivity anisotropy not concluded at the calculations. The comparison of FA values showed that there is a significant statistical difference between the mean FA value of tumor and normal soft tissues (P<0.05). DTI is a feasible technique for the assessment of electrical conductivity anisotropy of tissues.  It is crucial to quantify the electrical conductivity anisotropy data of tissues for numerical modeling of electroporation treatments.

Development of plasmid vector and electroporation condition for gene transfer in sporogenic lactic acid bacterium, Bacillus coagulans.

PubMed

Rhee, Mun Su; Kim, Jin-Woo; Qian, Yilei; Ingram, L O; Shanmugam, K T

2007-07-01

Bacillus coagulans is a sporogenic lactic acid bacterium that ferments glucose and xylose, major components of plant biomass, a potential feedstock for cellulosic ethanol. The temperature and pH for optimum rate of growth of B. coagulans (50 to 55 degrees C, pH 5.0) are very similar to that of commercially developed fungal cellulases (50 degrees C; pH 4.8). Due to this match, simultaneous saccharification and fermentation (SSF) of cellulose to products by B. coagulans is expected to require less cellulase than needed if the SSF is conducted at a sub-optimal temperature, such as 30 degrees C, the optimum for yeast, the main biocatalyst used by the ethanol industry. To fully exploit B. coagulans as a platform organism, we have developed an electroporation method to transfer plasmid DNA into this genetically recalcitrant bacterium. We also constructed a B. coagulans/E. coli shuttle vector, plasmid pMSR10 that contains the rep region from a native plasmid (pMSR0) present in B. coagulans strain P4-102B. The native plasmid, pMSR0 (6823bp), has 9 ORFs, and replicates by rolling-circle mode of replication. Plasmid pNW33N, developed for Geobacillus stearothermophilus, was also transformed into this host and stably maintained while several other Bacillus/Escherichia coli shuttle vector plasmids were not transformed into B. coagulans. The transformation efficiency of B. coagulans strain P4-102B using the plasmids pNW33N or pMSR10 was about 1.5x10(16) per mole of DNA. The availability of shuttle vectors and an electroporation method is expected to aid in genetic and metabolic engineering of B. coagulans.

An Electroacoustic Hearing Protector Simulator That Accurately Predicts Pressure Levels in the Ear Based on Standard Performance Metrics

DTIC Science & Technology

2013-08-01

earplug and earmuff showing HPD simulator elements for energy flow paths...unprotected or protected ear traditionally start with analysis of energy flow through schematic diagrams based on electroacoustic (EA) analogies between...Schröter, 1983; Schröter and Pösselt, 1986; Shaw and Thiessen, 1958, 1962; Zwislocki, 1957). The analysis method tracks energy flow through fluid and

Nonlinear storage models of unconfined flow through a shallow aquifer on an inclined base and their quasi-steady flow application

NASA Astrophysics Data System (ADS)

Varvaris, Ioannis; Gravanis, Elias; Koussis, Antonis; Akylas, Evangelos

2013-04-01

Hillslope processes involving flow through an inclined shallow aquifer range from subsurface stormflow to stream base flow (drought flow, or groundwater recession flow). In the case of recharge, the infiltrating water moves vertically as unsaturated flow until it reaches the saturated groundwater, where the flow is approximately parallel to the base of the aquifer. Boussinesq used the Dupuit-Forchheimer (D-F) hydraulic theory to formulate unconfined groundwater flow through a soil layer resting on an impervious inclined bed, deriving a nonlinear equation for the flow rate that consists of a linear gravity-driven component and a quadratic pressure-gradient component. Inserting that flow rate equation into the differential storage balance equation (volume conservation) Boussinesq obtained a nonlinear second-order partial differential equation for the depth. So far however, only few special solutions have been advanced for that governing equation. The nonlinearity of the equation of Boussinesq is the major obstacle to deriving a general analytical solution for the depth profile of unconfined flow on a sloping base with recharge (from which the discharges could be then determined). Henderson and Wooding (1964) were able to obtain an exact analytical solution for steady unconfined flow on a sloping base, with recharge, and their work deserves special note in the realm of solutions of the nonlinear equation of Boussinesq. However, the absence of a general solution for the transient case, which is of practical interest to hydrologists, has been the motivation for developing approximate solutions of the non-linear equation of Boussinesq. In this work, we derive the aquifer storage function by integrating analytically over the aquifer base the depth profiles resulting from the complete nonlinear Boussinesq equation for steady flow. This storage function consists of a linear and a nonlinear outflow-dependent term. Then, we use this physics-based storage function in the transient storage balance over the hillslope, obtaining analytical solutions of the outflow and the storage, for recharge and drainage, via a quasi-steady flow calculation. The hydraulically derived storage model is thus embedded in a quasi-steady approximation of transient unconfined flow in sloping aquifers. We generalise this hydrologic model of groundwater flow by modifying the storage function to be the weighted sum of the linear and the nonlinear storage terms, determining the weighting factor objectively from a known integral quantity of the flow (either an initial volume of water stored in the aquifer or a drained water volume). We demonstrate the validity of this model through comparisons with experimental data and simulation results.

Electroporation and use of hepatitis B virus envelope L proteins as bionanocapsules.

PubMed

Yamada, Tadanori; Jung, Joohee; Seno, Masaharu; Kondo, Akihiko; Ueda, Masakazu; Tanizawa, Katsuyuki; Kuroda, Shun'ichi

2012-06-01

Hepatitis B virus (HBV) envelope L proteins, when synthesized in yeast cells, form a hollow bionanocapsule (BNC) in which genes (including large plasmids up to 40 kbp), small interfering RNA (siRNA), drugs, and proteins can be enclosed by electroporation. BNCs made from L proteins have several advantages as a delivery system: Because they display a human liver-specific receptor (the pre-S region of the L protein) on their surface, BNCs can efficiently and specifically deliver their contents to human liver-derived cells and tissues ex vivo (in cell culture) and in vivo (in a mouse xenograft model). Retargeting can be achieved simply by substituting other biorecognition molecules such as antibodies, ligands, receptors, and homing peptides for the pre-S region. In addition, BNCs have already been proven to be safe for use in humans during their development as an immunogen of hepatitis B vaccine. This protocol describes the loading of BNCs and their use in cell culture and in vivo.

Time domain dielectric spectroscopy of nanosecond pulsed electric field induced changes in dielectric properties of pig whole blood.

PubMed

Zhuang, Jie; Kolb, Juergen F

2015-06-01

The dielectric spectra of fresh pig whole blood in the β-dispersion range after exposure to 300-nanosecond pulsed electric fields (nsPEFs) with amplitude higher than the supra-electroporation threshold for erythrocytes were recorded by time domain reflectometry dielectric spectroscopy. The implications of the dielectric parameters on the dynamics of post-pulse pore development were discussed in light of the Cole-Cole relaxation model. The temporal development of the Cole-Cole parameters indicates that nsPEFs induced significant poration and swelling of erythrocytes within the first 5 min. The results also show that the majority of erythrocytes could not fully recover from supra-electroporation up to 30 min. The findings of this study suggest that time domain dielectric spectroscopy is a promising label-free and real-time physiological measuring technique for nsPEF-blood related biomedical applications, capable of following the conformational and morphological changes of cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Polymer multilayer tattooing for enhanced DNA vaccination

PubMed Central

DeMuth, Peter C.; Min, Younjin; Huang, Bonnie; Kramer, Joshua A.; Miller, Andrew D.; Barouch, Dan H.; Hammond, Paula T.; Irvine, Darrell J.

2014-01-01

DNA vaccines have many potential benefits but have failed to generate robust immune responses in humans. Recently, methods such as in vivo electroporation have demonstrated improved performance, but an optimal strategy for safe, reproducible, and pain-free DNA vaccination remains elusive. Here we report an approach for rapid implantation of vaccine-loaded polymer films carrying DNA, immune-stimulatory RNA, and biodegradable polycations into the immune-cell-rich epidermis, using microneedles coated with releasable polyelectrolyte multilayers. Films transferred into the skin following brief microneedle application promoted local transfection and controlled the persistence of DNA and adjuvants in the skin from days to weeks, with kinetics determined by the film composition. These “multilayer tattoo” DNA vaccines induced immune responses against a model HIV antigen comparable to electroporation in mice, enhanced memory T-cell generation, and elicited 140-fold higher gene expression in non-human primate skin than intradermal DNA injection, indicating the potential of this strategy for enhancing DNA vaccination. PMID:23353628

Codon-optimized filovirus DNA vaccines delivered by intramuscular electroporation protect cynomolgus macaques from lethal Ebola and Marburg virus challenges.

PubMed

Grant-Klein, Rebecca J; Altamura, Louis A; Badger, Catherine V; Bounds, Callie E; Van Deusen, Nicole M; Kwilas, Steven A; Vu, Hong A; Warfield, Kelly L; Hooper, Jay W; Hannaman, Drew; Dupuy, Lesley C; Schmaljohn, Connie S

2015-01-01

Cynomolgus macaques were vaccinated by intramuscular electroporation with DNA plasmids expressing codon-optimized glycoprotein (GP) genes of Ebola virus (EBOV) or Marburg virus (MARV) or a combination of codon-optimized GP DNA vaccines for EBOV, MARV, Sudan virus and Ravn virus. When measured by ELISA, the individual vaccines elicited slightly higher IgG responses to EBOV or MARV than did the combination vaccines. No significant differences in immune responses of macaques given the individual or combination vaccines were measured by pseudovirion neutralization or IFN-γ ELISpot assays. Both the MARV and mixed vaccines were able to protect macaques from lethal MARV challenge (5/6 vs. 6/6). In contrast, a greater proportion of macaques vaccinated with the EBOV vaccine survived lethal EBOV challenge in comparison to those that received the mixed vaccine (5/6 vs. 1/6). EBOV challenge survivors had significantly higher pre-challenge neutralizing antibody titers than those that succumbed.

Applications of Tol2 Transposon-Mediated Gene Transfer for Stable Integration and Conditional Expression of Electroporated Genes in Chicken Embryos

NASA Astrophysics Data System (ADS)

Sato, Yuki; Takahashi, Yoshiko

Because of the high accessibility to developing embryos, avian embryos (chicken and quail) have long been used as a good model animal to study embryogenesis in vertebrates, especially amniotes (reviewed in Wolpert, 2004). The techniques used for “classical” avian embryology included tissue transplantations, tissue ablations, and cell-labeling by vital dye. At the end of the last century, the in ovo electropora tion technique was developed by Nakamura and his colleagues, and this modern method opened a way to study the roles of developmental genes directly in living embryos (Funahashi et al., 1999) reviewed in (Nakamura et al., 2004; Yasuda et al., 2000; Yasugi and Nakamura, 2000). This powerful technique allows us to introduce genes (DNA, RNA, morpholino) into embryos in a tissue-specific way by targeting a restricted area of embryonic tissues. Thus, the electroporation technique using chickens has provided numerous novel insights into the understanding of early development in vertebrates, making the chicken a unique model animal.

Cell Membrane Electropulsation: Chemical Analysis of Cell Membrane Modifications and Associated Transport Mechanisms.

PubMed

Azan, Antoine; Gailliègue, Florian; Mir, Lluis M; Breton, Marie

2017-01-01

The transport of substances across the cell membrane is complex because the main physiological role of the membrane is the control of the substances that would enter or exit the cells. Life would not be possible without this control. Cell electropulsation corresponds to the delivery of electric pulses to the cells and comprises cell electroporation and cell electropermeabilization. Cell electropulsation allows for the transport of non-permeant molecules across the membrane, bypassing the physiological limitations. In this chapter we discuss the changes occurring in the cell membrane during electroporation or electropermeabilization as they allow to understand which molecules can be transported as well as when and how their transport can occur. Electrophoretic or diffusive transports across the cell membrane can be distinguished. This understanding has a clear impact on the choice of the electrical parameters to be applied to the cells as well as on other aspects of the experimental protocols that have to be set to load the cells with non-permeant molecules.

Electroinduced Delivery of Hydrogel Nanoparticles in Colon 26 Cells, Visualized by Confocal Fluorescence System.

PubMed

Atanasova, Severina; Nikolova, Biliana; Murayama, Shuhei; Stoyanova, Elena; Tsoneva, Iana; Zhelev, Zhivko; Aoki, Ichio; Bakalova, Rumiana

2016-09-01

Nano-scale drug delivery systems (nano-DDS) are under intense investigation. Nano-platforms are developed for specific administration of small molecules, drugs, genes, contrast agents [quantum dots (QDs)] both in vivo and in vitro. Electroporation is a biophysical phenomenon which consists of the application of external electrical pulses across the cell membrane. The aim of this study was to research electro-assisted Colon 26 cell line internalization of QDs and QD-loaded nano-hydrogels (polymersomes) visualized by confocal microscopy and their influence on cell viability. The experiments were performed on the Colon 26 cancer cell line, using a confocal fluorescent imaging system and cell viability test. Electroporation facilitated the delivery of nanoparticles in vivo. We demonstrated increased voltage-dependent delivery of nanoparticles into cells after electrotreatment, without significant cell viability reduction. The delivery and retention of the polymersomes in vitro is a promising tool for future cancer treatment strategies and nanomedcine. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Transformation of Epichloë typhina by electroporation of conidia

PubMed Central

2011-01-01

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. PMID:21375770

Angiography in the Isolated Perfused Kidney: Radiological Evaluation of Vascular Protection in Tissue Ablation by Nonthermal Irreversible Electroporation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wendler, Johann Jakob, E-mail: johann.wendler@med.ovgu.de; Pech, Maciej; Blaschke, Simon

2012-04-15

Purpose: The nonthermal irreversible electroporation (NTIRE) is a novel nonthermal tissue ablation technique by local application of high-voltage current within microseconds leading to a delayed apoptosis. The purpose of this experimental study was the first angiographic evaluation of the acute damage of renal vascular structure in NTIRE. Methods: Results of conventional dynamic digital substraction angiography (DSA) and visualization of the terminal vascular bed of renal parenchyma by high-resolution X-ray in mammography technique were evaluated before, during, and after NTIRE of three isolated perfused porcine ex vivo kidneys. Results: In the dedicated investigation, no acute vascular destruction of the renal parenchymamore » and no dysfunction of the kidney perfusion model were observed during or after NTIRE. Conspicuous were concentric wave-like fluctuations of the DSA contrast agent simultaneous to the NTIRE pulses resulting from NTIRE pulse shock wave. Conclusion: The NTIRE offers an ablation method with no acute collateral vascular damage in angiographic evaluation.« less

Ultra-localized single cell electroporation using silicon nanowires.

PubMed

Jokilaakso, Nima; Salm, Eric; Chen, Aaron; Millet, Larry; Guevara, Carlos Duarte; Dorvel, Brian; Reddy, Bobby; Karlstrom, Amelie Eriksson; Chen, Yu; Ji, Hongmiao; Chen, Yu; Sooryakumar, Ratnasingham; Bashir, Rashid

2013-02-07

Analysis of cell-to-cell variation can further the understanding of intracellular processes and the role of individual cell function within a larger cell population. The ability to precisely lyse single cells can be used to release cellular components to resolve cellular heterogeneity that might be obscured when whole populations are examined. We report a method to position and lyse individual cells on silicon nanowire and nanoribbon biological field effect transistors. In this study, HT-29 cancer cells were positioned on top of transistors by manipulating magnetic beads using external magnetic fields. Ultra-rapid cell lysis was subsequently performed by applying 600-900 mV(pp) at 10 MHz for as little as 2 ms across the transistor channel and the bulk substrate. We show that the fringing electric field at the device surface disrupts the cell membrane, leading to lysis from irreversible electroporation. This methodology allows rapid and simple single cell lysis and analysis with potential applications in medical diagnostics, proteome analysis and developmental biology studies.

Flow through a very porous obstacle in a shallow channel.

PubMed

Creed, M J; Draper, S; Nishino, T; Borthwick, A G L

2017-04-01

A theoretical model, informed by numerical simulations based on the shallow water equations, is developed to predict the flow passing through and around a uniform porous obstacle in a shallow channel, where background friction is important. This problem is relevant to a number of practical situations, including flow through aquatic vegetation, the performance of arrays of turbines in tidal channels and hydrodynamic forces on offshore structures. To demonstrate this relevance, the theoretical model is used to (i) reinterpret core flow velocities in existing laboratory-based data for an array of emergent cylinders in shallow water emulating aquatic vegetation and (ii) reassess the optimum arrangement of tidal turbines to generate power in a tidal channel. Comparison with laboratory-based data indicates a maximum obstacle resistance (or minimum porosity) for which the present theoretical model is valid. When the obstacle resistance is above this threshold the shallow water equations do not provide an adequate representation of the flow, and the theoretical model over-predicts the core flow passing through the obstacle. The second application of the model confirms that natural bed resistance increases the power extraction potential for a partial tidal fence in a shallow channel and alters the optimum arrangement of turbines within the fence.

The Effect of Voltage Charging on the Transport Properties of Gold Nanotube Membranes.

PubMed

Experton, Juliette; Martin, Charles R

2018-05-01

Porous membranes are used in chemical separations and in many electrochemical processes and devices. Research on the transport properties of a unique class of porous membranes that contain monodisperse gold nanotubes traversing the entire membrane thickness is reviewed here. These gold nanotubes can act as conduits for ionic and molecular transports through the membrane. Because the tubes are electronically conductive, they can be electrochemically charged by applying a voltage to the membrane. How this "voltage charging" affects the transport properties of gold nanotube membranes is the subject of this Review. Experiments showing that voltage charging can be used to reversibly switch the membrane between ideally cation- and anion-transporting states are reviewed. Voltage charging can also be used to enhance the ionic conductivity of gold nanotube membranes. Finally, voltage charging to accomplish electroporation of living bacteria as they pass through gold nanotube membranes is reviewed. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effective gene delivery to Trypanosoma cruzi epimastigotes through nucleofection.

PubMed

Pacheco-Lugo, Lisandro; Díaz-Olmos, Yirys; Sáenz-García, José; Probst, Christian Macagnan; DaRocha, Wanderson Duarte

2017-06-01

New opportunities have raised to study the gene function approaches of Trypanosoma cruzi after its genome sequencing in 2005. Functional genomic approaches in Trypanosoma cruzi are challenging due to the reduced tools available for genetic manipulation, as well as to the reduced efficiency of the transient transfection conducted through conventional methods. The Amaxa nucleofector device was systematically tested in the present study in order to improve the electroporation conditions in the epimastigote forms of T. cruzi. The transfection efficiency was quantified using the green fluorescent protein (GFP) as reporter gene followed by cell survival assessment. The herein used nucleofection parameters have increased the survival rates (>90%) and the transfection efficiency by approximately 35%. The small amount of epimastigotes and DNA required for the nucleofection can turn the method adopted here into an attractive tool for high throughput screening (HTS) applications, and for gene editing in parasites where genetic manipulation tools remain relatively scarce. Copyright © 2017 Elsevier B.V. All rights reserved.

Microcapillary-Based Flow-Through Immunosensor and Displacement Immunoassay Using the Same.

DTIC Science & Technology

1997-04-28

an antibody. If desired, an electroosmotic 24 pump may be used to flow fluid through the microcapillary or 25 microcapillaries in the chip...8 for field use. 9 Fig. 1C shows a flow immunosensor chip 100. Buffer flow 10 through microcapillary passage 102 by virtue of an electroosmotic ...Power for an 23 electroosmotic pump or other fluid pump, as well as any other on- 24 chip components, may be provided by a battery incorporated into

Site-specific integration of Streptomyces PhiC31 integrase-based vectors in the chromosome of Rhodococcus equi.

PubMed

Hong, Yang; Hondalus, Mary K

2008-10-01

Streptomyces PhiC31-based site-specific integration was used to transform the facultative intracellular pathogen Rhodococcus equi. The transformation efficiency of vectors incorporating the PhiC31 integrase and attP sites was comparable to that of replication plasmids using the same electroporation procedure. A single attB integration site was identified within an ORF encoding a pirin-like protein, which deviates slightly from the consensus sequence of Streptomyces attB sites. Vector integration was stably maintained in the R. equi chromosome for as many as 100 generations during unselected passage in vitro. In addition, integration does not appear to affect the replication of bacteria inside macrophages. Finally, this integration system was also used to successfully complement an R. equi mutant.

Investigation and Verification of the Aerodynamic Performance of a Fan/Booster with Through-flow Method

NASA Astrophysics Data System (ADS)

Liu, Xiaoheng; Jin, Donghai; Gui, Xingmin

2018-04-01

Through-flow method is still widely applied in the revolution of the design of a turbomachinery, which can provide not merely the performance characteristic but also the flow field. In this study, a program based on the through-flow method was proposed, which had been verified by many other numerical examples. So as to improve the accuracy of the calculation, abundant loss and deviation models dependent on the real geometry of engine were put into use, such as: viscous losses, overflow in gaps, leakage from a flow path through seals. By means of this program, the aerodynamic performance of a certain high through-flow commercial fan/booster was investigated. On account of the radial distributions of the relevant parameters, flow deterioration in this machine was speculated. To confirm this surmise, 3-D numerical simulation was carried out with the help of the NUMECA software. Through detailed analysis, the speculation above was demonstrated, which provide sufficient evidence for the conclusion that the through-flow method is an essential and effective method for the performance prediction of the fan/booster.

Exogenous Gene Integration for Microalgal Cell Transformation Using a Nanowire-Incorporated Microdevice.

PubMed

Bae, Sunwoong; Park, Seunghye; Kim, Jung; Choi, Jong Seob; Kim, Kyung Hoon; Kwon, Donguk; Jin, EonSeon; Park, Inkyu; Kim, Do Hyun; Seo, Tae Seok

2015-12-16

Superior green algal cells showing high lipid production and rapid growth rate are considered as an alternative for the next generation green energy resources. To achieve the biomass based energy generation, transformed microalgae with superlative properties should be developed through genetic engineering. Contrary to the normal cells, microalgae have rigid cell walls, so that target gene delivery into cells is challengeable. In this study, we report a ZnO nanowire-incorporated microdevice for a high throughput microalgal transformation. The proposed microdevice was equipped with not only a ZnO nanowire in the microchannel for gene delivery into cells but also a pneumatic polydimethylsiloxane (PDMS) microvalve to modulate the cellular attachment and detachment from the nanowire. As a model, hygromycin B resistance gene cassette (Hyg3) was functionalized on the hydrothermally grown ZnO nanowires through a disulfide bond and released into green algal cells, Chlamydomonas reinhardtii, by reductive cleavage. During Hyg3 gene delivery, a monolithic PDMS membrane was bent down, so that algal cells were pushed down toward ZnO nanowires. The supply of vacuum in the pneumatic line made the PDMS membrane bend up, enabling the gene delivered algal cells to be recovered from the outlet of the microchannel. We successfully confirmed Hyg3 gene integrated in microalgae by amplifying the inserted gene through polymerase chain reaction (PCR) and DNA sequencing. The efficiency of the gene delivery to algal cells using the ZnO nanowire-incorporated microdevice was 6.52 × 10(4)- and 9.66 × 10(4)-fold higher than that of a traditional glass bead beating and electroporation.

Electric field-based technologies for valorization of bioresources.

PubMed

Rocha, Cristina M R; Genisheva, Zlatina; Ferreira-Santos, Pedro; Rodrigues, Rui; Vicente, António A; Teixeira, José A; Pereira, Ricardo N

2018-04-01

This review provides an overview of recent research on electrotechnologies applied to the valorization of bioresources. Following a comprehensive summary of the current status of the application of well-known electric-based processing technologies, such as pulsed electric fields (PEF) and high voltage electrical discharges (HVED), the application of moderate electric fields (MEF) as an extraction or valorization technology will be considered in detail. MEF, known by its improved energy efficiency and claimed electroporation effects (allowing enhanced extraction yields), may also originate high heating rates - ohmic heating (OH) effect - allowing thermal stabilization of waste stream for other added-value applications. MEF is a simple technology that mostly makes use of green solvents (mainly water) and that can be used on functionalization of compounds of biological origin broadening their application range. The substantial increase of MEF-based plants installed in industries worldwide suggests its straightforward application for waste recovery. Copyright © 2018 Elsevier Ltd. All rights reserved.

Flow through a very porous obstacle in a shallow channel

PubMed Central

Draper, S.; Nishino, T.; Borthwick, A. G. L.

2017-01-01

A theoretical model, informed by numerical simulations based on the shallow water equations, is developed to predict the flow passing through and around a uniform porous obstacle in a shallow channel, where background friction is important. This problem is relevant to a number of practical situations, including flow through aquatic vegetation, the performance of arrays of turbines in tidal channels and hydrodynamic forces on offshore structures. To demonstrate this relevance, the theoretical model is used to (i) reinterpret core flow velocities in existing laboratory-based data for an array of emergent cylinders in shallow water emulating aquatic vegetation and (ii) reassess the optimum arrangement of tidal turbines to generate power in a tidal channel. Comparison with laboratory-based data indicates a maximum obstacle resistance (or minimum porosity) for which the present theoretical model is valid. When the obstacle resistance is above this threshold the shallow water equations do not provide an adequate representation of the flow, and the theoretical model over-predicts the core flow passing through the obstacle. The second application of the model confirms that natural bed resistance increases the power extraction potential for a partial tidal fence in a shallow channel and alters the optimum arrangement of turbines within the fence. PMID:28484321

A comparative study on the transdermal penetration effect of gaseous and aqueous plasma reactive species

NASA Astrophysics Data System (ADS)

Liu, Xin; Gan, Lu; Ma, Mingyu; Zhang, Song; Liu, Jingjing; Chen, Hongxiang; Liu, Dawei; Lu, Xinpei

2018-02-01

To improve the depth of plasma active species in the skin, it is very important to develop skin disease treatment using plasma. In this article, an air plasma source was used to work directly with the skin of a mouse. A tortuous pathway, hair follicles, electroporation and a microneedle do not aid the transdermal delivery of gaseous plasma active species, therefore these gaseous plasma active species cannot penetrate mouse skin with a thickness of ~0.75 mm. The plasma activated water (PAW) produced by the air plasma source was used to study the transdermal penetration of the aqueous plasma activated species. This aqueous plasma activated species can penetrate the skin through hair follicles, intercellular and transcellular routes. The pH of the PAW did not affect the penetration efficiency of the aqueous plasma active species.

Effect of viscoelasticity on the flow pattern and the volumetric flow rate in electroosmotic flows through a microchannel.

PubMed

Park, H M; Lee, W M

2008-07-01

Many lab-on-a-chip based microsystems process biofluids such as blood and DNA solutions. These fluids are viscoelastic and show extraordinary flow behaviors, not existing in Newtonian fluids. Adopting appropriate constitutive equations these exotic flow behaviors can be modeled and predicted reasonably using various numerical methods. In the present paper, we investigate viscoelastic electroosmotic flows through a rectangular straight microchannel with and without pressure gradient. It is shown that the volumetric flow rates of viscoelastic fluids are significantly different from those of Newtonian fluids under the same external electric field and pressure gradient. Moreover, when pressure gradient is imposed on the microchannel there appear appreciable secondary flows in the viscoelastic fluids, which is never possible for Newtonian laminar flows through straight microchannels. The retarded or enhanced volumetric flow rates and secondary flows affect dispersion of solutes in the microchannel nontrivially.

Flow through the nasal cavity of the spiny dogfish, Squalus acanthias

NASA Astrophysics Data System (ADS)

Timm-Davis, L. L.; Fish, F. E.

2015-12-01

The nasal cavity of spiny dogfish is a blind capsule with no internal connection to the oral cavity. Water is envisioned to flow through the cavity in a smooth, continuous flow pattern; however, this assumption is based on previous descriptions of the morphology of the olfactory cavity. No experimentation on the flow through the internal nasal cavity has been reported. Morphology of the head of the spiny dogfish ( Squalus acanthias) does not suggest a close external connection between the oral and nasal systems. However, dye visualization showed that there was flow through the nasal apparatus and from the excurrent nostril to the mouth when respiratory flows were simulated. The hydrodynamic flow through the nasal cavity was observed from flow tank experiments. The dorsum of the nasal cavity of shark heads from dead animals was exposed by dissection and a glass plate was glued over of the exposed cavity. When the head was placed in a flow, dye was observed to be drawn passively into the cavity showing a complex, three-dimensional hydrodynamic flow. Dye entered the incurrent nostril, flowed through the nasal lamellae, crossed over and under the nasal valve, and circulated around the nasal valve before exiting the excurrent nostril. When the nasal valve was removed, the dye became stagnant and back flowed out through the incurrent nostril. The single nasal valve has a hydrodynamic function that organizes a coherent flow of water through the cavity without disruption. The results suggest that the morphology of the nasal apparatus in concert with respiratory flow and ambient flows from active swimming can be used to draw water through the olfactory cavity of the shark.

Enabling skin vaccination using new delivery technologies

PubMed Central

Kim, Yeu-Chun; Prausnitz, Mark R.

2011-01-01

The skin is known to be a highly immunogenic site for vaccination, but few vaccines in clinical use target skin largely because conventional intradermal injection is difficult and unreliable to perform. Now, a number of new or newly adapted delivery technologies have been shown to administer vaccine to the skin either by non-invasive or minimally invasive methods. Non-invasive methods include high-velocity powder and liquid jet injection, as well as diffusion-based patches in combination with skin abrasion, thermal ablation, ultrasound, electroporation, and chemical enhancers. Minimally invasive methods are generally based on small needles, including solid microneedle patches, hollow microneedle injections, and tattoo guns. The introduction of these advanced delivery technologies can make the skin a site for simple, reliable vaccination that increases vaccine immunogenicity and offers logistical advantages to improve the speed and coverage of vaccination. PMID:21799951

Enabling skin vaccination using new delivery technologies

PubMed Central

Kim, Yeu-Chun; Prausnitz, Mark R.

2011-01-01

The skin is known to be a highly immunogenic site for vaccination, but few vaccines in clinical use target skin largely because conventional intradermal injection is difficult and unreliable to perform. Now, a number of new or newly adapted delivery technologies have been shown to administer vaccine to the skin either by non-invasive or minimally invasive methods. Non-invasive methods include high-velocity powder and liquid jet injection, as well as diffusion-based patches in combination with skin abrasion, thermal ablation, ultrasound, electroporation, and chemical enhancers. Minimally invasive methods are generally based on small needles, including solid microneedle patches, hollow microneedle injections and tattoo guns. The introduction of these advanced delivery technologies can make the skin a site for simple, reliable vaccination that increases vaccine immunogenicity and offers logistical advantages to improve the speed and coverage of vaccination. PMID:21472533

Anticancer Therapeutic Potential of VEGI, an Antiangiogenic Cytokine

DTIC Science & Technology

2002-10-01

PCR product. The blots were hybridized at 42°C overnight and washed in wash buffer 1 ( 2 x SSC, 0.1% sodium lauryl sulfate ) and wash buffer 2 (1 x SSC...transfected into MDA- MIB-231 breast cancer cells by electroporation. Stable transfectants were selected in 2 mg/ml G418 sulfate (Invitrogen). Conditioned

Construction and symbiotic competence of a luxA-deletion mutant of Vibrio fischeri.

PubMed

Visick, K G; Ruby, E G

1996-10-10

Bioluminescence by the squid Euprymna scolopes requires colonization of its light organ by the symbiotic luminous bacterium Vibrio fischeri. Investigation of the genetic determinants underlying bacterial symbiotic competence in this system has necessitated the continuing establishment and application of molecular genetic techniques in V. fischeri. We developed a procedure for the introduction of plasmid DNA into V. fischeri by electroporation, and isolated a mutant strain that overcame the apparent restriction barrier between V. fischeri and Escherichia coli. Using the technique of electroporation in combination with that of gene replacement, we constructed a non-luminous strain of V. fischeri (delta luxA::erm). In addition, we used the transducing phage rp-1 for the first time to transfer a chromosomal antibiotic resistance marker to another strain of V. fischeri. The luxA mutant was able to colonize E. scolopes as quickly and to the same extent as wild type. This result suggested that, at least during the initial stages of colonization, luminescence per se is not an essential factor for the symbiotic infection.

Systems for column-based separations, methods of forming packed columns, and methods of purifying sample components

DOEpatents

Egorov, Oleg B.; O'Hara, Matthew J.; Grate, Jay W.; Chandler, Darrell P.; Brockman, Fred J.; Bruckner-Lea, Cynthia J.

2000-01-01

The invention encompasses systems for column-based separations, methods of packing and unpacking columns and methods of separating components of samples. In one aspect, the invention includes a method of packing and unpacking a column chamber, comprising: a) packing a matrix material within a column chamber to form a packed column; and b) after the packing, unpacking the matrix material from the column chamber without moving the column chamber. In another aspect, the invention includes a system for column-based separations, comprising: a) a fluid passageway, the fluid passageway comprising a column chamber and a flow path in fluid communication with the column chamber, the flow path being obstructed by a retaining material permeable to a carrier fluid and impermeable to a column matrix material suspended in the carrier fluid, the flow path extending through the column chamber and through the retaining material, the flow path being configured to form a packed column within the column chamber when a suspension of the fluid and the column matrix material is flowed along the flow path; and b) the fluid passageway extending through a valve intermediate the column chamber and the retaining material.

Systems For Column-Based Separations, Methods Of Forming Packed Columns, And Methods Of Purifying Sample Components

DOEpatents

Egorov, Oleg B.; O'Hara, Matthew J.; Grate, Jay W.; Chandler, Darrell P.; Brockman, Fred J.; Bruckner-Lea, Cynthia J.

2006-02-21

The invention encompasses systems for column-based separations, methods of packing and unpacking columns and methods of separating components of samples. In one aspect, the invention includes a method of packing and unpacking a column chamber, comprising: a) packing a matrix material within a column chamber to form a packed column; and b) after the packing, unpacking the matrix material from the column chamber without moving the column chamber. In another aspect, the invention includes a system for column-based separations, comprising: a) a fluid passageway, the fluid passageway comprising a column chamber and a flow path in fluid communication with the column chamber, the flow path being obstructed by a retaining material permeable to a carrier fluid and impermeable to a column matrix material suspended in the carrier fluid, the flow path extending through the column chamber and through the retaining material, the flow path being configured to form a packed column within the column chamber when a suspension of the fluid and the column matrix material is flowed along the flow path; and b) the fluid passageway extending through a valve intermediate the column chamber and the retaining material.

Systems For Column-Based Separations, Methods Of Forming Packed Columns, And Methods Of Purifying Sample Components.

DOEpatents

Egorov, Oleg B.; O'Hara, Matthew J.; Grate, Jay W.; Chandler, Darrell P.; Brockman, Fred J.; Bruckner-Lea, Cynthia J.

2004-08-24

The invention encompasses systems for column-based separations, methods of packing and unpacking columns and methods of separating components of samples. In one aspect, the invention includes a method of packing and unpacking a column chamber, comprising: a) packing a matrix material within a column chamber to form a packed column; and b) after the packing, unpacking the matrix material from the column chamber without moving the column chamber. In another aspect, the invention includes a system for column-based separations, comprising: a) a fluid passageway, the fluid passageway comprising a column chamber and a flow path in fluid communication with the column chamber, the flow path being obstructed by a retaining material permeable to a carrier fluid and impermeable to a column matrix material suspended in the carrier fluid, the flow path extending through the column chamber and through the retaining material, the flow path being configured to form a packed column within the column chamber when a suspension of the fluid and the column matrix material is flowed along the flow path; and b) the fluid passageway extending through a valve intermediate the column chamber and the retaining material.

Development of an efficient, non-viral transfection method for studying gene function and bone growth in human primary cranial suture mesenchymal cells reveals that the cells respond to BMP2 and BMP3.

PubMed

Dwivedi, Prem P; Anderson, Peter J; Powell, Barry C

2012-08-03

Achieving efficient introduction of plasmid DNA into primary cultures of mammalian cells is a common problem in biomedical research. Human primary cranial suture cells are derived from the connective mesenchymal tissue between the bone forming regions at the edges of the calvarial plates of the skull. Typically they are referred to as suture mesenchymal cells and are a heterogeneous population responsible for driving the rapid skull growth that occurs in utero and postnatally. To better understand the molecular mechanisms involved in skull growth, and in abnormal growth conditions, such as craniosynostosis, caused by premature bony fusion, it is essential to be able to easily introduce genes into primary bone forming cells to study their function. A comparison of several lipid-based techniques with two electroporation-based techniques demonstrated that the electroporation method known as nucleofection produced the best transfection efficiency. The parameters of nucleofection, including cell number, amount of DNA and nucleofection program, were optimized for transfection efficiency and cell survival. Two different genes and two promoter reporter vectors were used to validate the nucleofection method and the responses of human primary suture mesenchymal cells by fluorescence microscopy, RT-PCR and the dual luciferase assay. Quantification of bone morphogenetic protein (BMP) signalling using luciferase reporters demonstrated robust responses of the cells to both osteogenic BMP2 and to the anti-osteogenic BMP3. A nucleofection protocol has been developed that provides a simple and efficient, non-viral alternative method for in vitro studies of gene and protein function in human skull growth. Human primary suture mesenchymal cells exhibit robust responses to BMP2 and BMP3, and thus nucleofection can be a valuable method for studying the potential competing action of these two bone growth factors in a model system of cranial bone growth.

Specification of spatial identities of cerebellar neuron progenitors by ptf1a and atoh1 for proper production of GABAergic and glutamatergic neurons.

PubMed

Yamada, Mayumi; Seto, Yusuke; Taya, Shinichiro; Owa, Tomoo; Inoue, Yukiko U; Inoue, Takayoshi; Kawaguchi, Yoshiya; Nabeshima, Yo-Ichi; Hoshino, Mikio

2014-04-02

In the cerebellum, the bHLH transcription factors Ptf1a and Atoh1 are expressed in distinct neuroepithelial regions, the ventricular zone (VZ) and the rhombic lip (RL), and are required for producing GABAergic and glutamatergic neurons, respectively. However, it is unclear whether Ptf1a or Atoh1 is sufficient for specifying GABAergic or glutamatergic neuronal fates. To test this, we generated two novel knock-in mouse lines, Ptf1a(Atoh1) and Atoh1(Ptf1a), that are designed to express Atoh1 and Ptf1a ectopically in the VZ and RL, respectively. In Ptf1a(Atoh1) embryos, ectopically Atoh1-expressing VZ cells produced glutamatergic neurons, including granule cells and deep cerebellar nuclei neurons. Correspondingly, in Atoh1(Ptf1a) animals, ectopically Ptf1a-expressing RL cells produced GABAergic populations, such as Purkinje cells and GABAergic interneurons. Consistent results were also obtained from in utero electroporation of Ptf1a or Atoh1 into embryonic cerebella, suggesting that Ptf1a and Atoh1 are essential and sufficient for GABAergic versus glutamatergic specification in the neuroepithelium. Furthermore, birthdating analyses with BrdU in the knock-in mice or with electroporation studies showed that ectopically produced fate-changed neuronal types were generated at temporal schedules closely simulating those of the wild-type RL and VZ, suggesting that the VZ and RL share common temporal information. Observations of knock-in brains as well as electroporated brains revealed that Ptf1a and Atoh1 mutually negatively regulate their expression, probably contributing to formation of non-overlapping neuroepithelial domains. These findings suggest that Ptf1a and Atoh1 specify spatial identities of cerebellar neuron progenitors in the neuroepithelium, leading to appropriate production of GABAergic and glutamatergic neurons, respectively.

Towards the creation of decellularized organ constructs using irreversible electroporation and active mechanical perfusion

PubMed Central

2010-01-01

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 parameters such as pulse length, repetition rate, and field strength on whole organ structures. PMID:21143979

Age-dependent Impairment of HIF-1α̣Expression in Diabetic Mice: Correction with Electroporation-facilitated Gene Therapy Increases Wound Healing, Angiogenesis, and Circulating Angiogenic Cells

PubMed Central

Liu, Lixin; Marti, Guy P.; Wei, Xiaofei; Zhang, Xianjie; Zhang, Huafeng; Liu, Ye V.; Nastai, Manuel; Semenza, Gregg L.; Harmon, John W.

2009-01-01

Wound healing is impaired in elderly patients with diabetes mellitus. We hypothesized that age-dependent impairment of cutaneous wound healing in db/db diabetic mice: (a) would correlate with reduced expression of the transcription factor hypoxia-inducible factor 1α (HIF-1α) as well as its downstream target genes; and (b) could be overcome by HIF-1α replacement therapy. Wound closure, angiogenesis, and mRNA expression in excisional skin wounds were analyzed and circulating angiogenic cells were quantified in db/db mice that were untreated or received electroporation-facilitated HIF-1α gene therapy. HIF-1α mRNA levels in wound tissue were significantly reduced in older (4–6 months) as compared to younger (1.5–2 months) db/db mice. Expression of mRNAs encoding the angiogenic cytokines vascular endothelial growth factor (VEGF), angiopoietin 1 (ANGPT1), ANGPT2, platelet derived growth factor B (PDGF-B), and placental growth factor (PLGF) was also impaired in wounds of older db/db mice. Intradermal injection of plasmid gWIZ-CA5, which encodes a constitutively active form of HIF-1α, followed by electroporation, induced increased levels of HIF-1α mRNA at the injection site on day 3 and increased levels of VEGF, PLGF, PDGF-B, and ANGPT2 mRNA on day 7. Circulating angiogenic cells in peripheral blood increased 10-fold in mice treated with gWIZ-CA5. Wound closure was significantly accelerated in db/db mice treated with gWIZ-CA5 as compared to mice treated with empty vector. Thus, HIF-1α gene therapy corrects the age-dependent impairment of HIF-1α expression, angiogenic cytokine expression, and circulating angiogenic cells that contribute to the age-dependent impairment of wound healing in db/db mice. PMID:18506785

T-cell receptor transfer for boosting HIV-1-specific T-cell immunity in HIV-1-infected patients.

PubMed

Mummert, Christiane; Hofmann, Christian; Hückelhoven, Angela G; Bergmann, Silke; Mueller-Schmucker, Sandra M; Harrer, Ellen G; Dörrie, Jan; Schaft, Niels; Harrer, Thomas

2016-09-10

Strategies to cure HIV-1 infection require the eradication of viral reservoirs. An innovative approach for boosting the cytotoxic T-lymphocyte response is the transfer of T-cell receptors (TCRs). Previously, we have shown that electroporation of TCR-encoding mRNA is able to reprogram CD8 T cells derived from healthy donors. So far, it is unknown whether the transfer of HIV-1-specific TCRs is capable to reprogram CD8 T cells of HIV-1-infected patients. To assess the efficiency of TCR-transfer by mRNA electroporation and the functionality of reprogramed T cells in HIV-1-infected patients, we performed an in-vitro analysis of TCR-transfer into T cells from HIV-1-infected patients in various stages of disease and from healthy controls. Peripheral blood mononuclear cells from 16 HIV-1-infected patients (nine HLA-A02-positive, seven HLA-A02-negative) and from five healthy controls were electroporated with mRNA-constructs encoding TCRs specific for the HLA-A02/HIV-1-gag p17 epitope SLYNTVATL (SL9). Functionality of the TCRs was measured by γIFN-ELISpot assays. SL9/TCR transfection into peripheral blood mononuclear cells from both HLA-A02-positive and HLA-A02-negative HIV-1-infected patients and from healthy blood donors reprogramed T cells for recognition of SL9-presenting HLA-A02-positive cells in γIFN-ELISpot assays. SL9/TCR-transfer into T cells from an immunodeficient AIDS patient could induce recognition of SL9-expressing target cells only after reversion of T-cell dysfunction by antiretroviral therapy. The transfer of HIV-1-p17-specific TCRs into T cells is functional both in HIV-1-infected patients as well as in healthy blood donors. TCR-transfer is a promising method to boost the immune system against HIV-1.

A calculation procedure for viscous flow in turbomachines, volume 3. [computer programs

NASA Technical Reports Server (NTRS)

Khalil, I.; Sheoran, Y.; Tabakoff, W.

1980-01-01

A method for analyzing the nonadiabatic viscous flow through turbomachine blade passages was developed. The field analysis is based upon the numerical integration of the full incompressible Navier-Stokes equations, together with the energy equation on the blade-to-blade surface. A FORTRAN IV computer program was written based on this method. The numerical code used to solve the governing equations employs a nonorthogonal boundary fitted coordinate system. The flow may be axial, radial or mixed and there may be a change in stream channel thickness in the through-flow direction. The inputs required for two FORTRAN IV programs are presented. The first program considers laminar flows and the second can handle turbulent flows. Numerical examples are included to illustrate the use of the program, and to show the results that are obtained.

Testing random forest classification for identifying lava flows and mapping age groups on a single Landsat 8 image

NASA Astrophysics Data System (ADS)

Li, Long; Solana, Carmen; Canters, Frank; Kervyn, Matthieu

2017-10-01

Mapping lava flows using satellite images is an important application of remote sensing in volcanology. Several volcanoes have been mapped through remote sensing using a wide range of data, from optical to thermal infrared and radar images, using techniques such as manual mapping, supervised/unsupervised classification, and elevation subtraction. So far, spectral-based mapping applications mainly focus on the use of traditional pixel-based classifiers, without much investigation into the added value of object-based approaches and into advantages of using machine learning algorithms. In this study, Nyamuragira, characterized by a series of > 20 overlapping lava flows erupted over the last century, was used as a case study. The random forest classifier was tested to map lava flows based on pixels and objects. Image classification was conducted for the 20 individual flows and for 8 groups of flows of similar age using a Landsat 8 image and a DEM of the volcano, both at 30-meter spatial resolution. Results show that object-based classification produces maps with continuous and homogeneous lava surfaces, in agreement with the physical characteristics of lava flows, while lava flows mapped through the pixel-based classification are heterogeneous and fragmented including much "salt and pepper noise". In terms of accuracy, both pixel-based and object-based classification performs well but the former results in higher accuracies than the latter except for mapping lava flow age groups without using topographic features. It is concluded that despite spectral similarity, lava flows of contrasting age can be well discriminated and mapped by means of image classification. The classification approach demonstrated in this study only requires easily accessible image data and can be applied to other volcanoes as well if there is sufficient information to calibrate the mapping.

Irreversible electroporation therapy in the management of locally advanced pancreatic adenocarcinoma.

PubMed

Martin, Robert C G; McFarland, Kelli; Ellis, Susan; Velanovich, Vic

2012-09-01

Locally advanced pancreatic cancer patients have limited options for disease control. Local ablation technologies based on thermal damage have been used but are associated with major complications in this region of the pancreas. Irreversible electroporation (IRE) is a nonthermal ablation technology that we have shown is safe near vital vascular and ductal structures. The aim of this study was to evaluate the safety and efficacy of IRE as a therapy in the treatment of locally advanced pancreatic cancer. We performed a prospective multi-institutional pilot evaluation of patients undergoing IRE for locally advanced pancreatic cancer from December 2009 to March 2011. These patients were evaluated for 90-day morbidity, mortality, and local disease control. Twenty-seven patients (13 women and 14 men) underwent IRE, with median age of 61 years (range 45 to 80 years). Eight patients underwent margin accentuation with IRE in combination with left-sided resection (n = 4) or pancreatic head resection (n = 4). Nineteen patients had in situ IRE. All patients underwent successful IRE, with intraoperative imaging confirming effective delivery of therapy. All 27 patients demonstrated nonclinically relevant elevation of their amylase and lipase, which peaked at 48 hours and returned to normal at 72 hour postprocedure. There has been one 90-day mortality. No patient has shown evidence of clinical pancreatitis or fistula formation. After all patients have completed 90-day follow-up, there has been 100% ablation success. IRE ablation of locally advanced pancreatic cancer tumors is a safe and feasible primary local treatment in unresectable, locally advanced disease. Confirming these early results must occur in a planned phase II investigational device exemption (IDE) study to be initiated in 2012. Copyright © 2012 American College of Surgeons. Published by Elsevier Inc. All rights reserved.

An improved Graves' disease model established by using in vivo electroporation exhibited long-term immunity to hyperthyroidism in BALB/c mice.

PubMed

Kaneda, Toshio; Honda, Asako; Hakozaki, Atsushi; Fuse, Tetsuya; Muto, Akihiro; Yoshida, Tadashi

2007-05-01

In Graves' disease, the overstimulation of the thyroid gland and hyperthyroidism are caused by autoantibodies directed against the TSH receptor (TSHR) that mimics the action of TSH. The establishment of an animal model is an important step to study the pathophysiology of autoimmune hyperthyroidism and for immunological analysis. In this study, we adopted the technique of electroporation (EP) for genetic immunization to achieve considerable enhancement of in vivo human TSHR (hTSHR) expression and efficient induction of hyperthyroidism in mice. In a preliminary study using beta-galactosidase (beta-gal) expression vectors, beta-gal introduced into the muscle by EP showed over 40-fold higher enzymatic activity than that introduced via previous direct gene transfer methods. The sustained hTSHR mRNA expression derived from cDNA transferred by EP was detectable in muscle tissue for at least 2 wk by RT-PCR. Based on these results, we induced hyperthyroidism via two expression vectors inserted with hTSHR or hTSHR289His cDNA. Consequently, 12.0-31.8% BALB/c mice immunized with hTSHR and 79.2-95.7% immunized with hTSHR289His showed high total T(4) levels due to the TSHR-stimulating antibody after three to four times repeated immunization by EP, and thyroid follicles of which were hyperplastic and had highly irregular epithelium. Moreover, TSHR-stimulating antibody surprisingly persisted more than 8 months after the last immunization. These results demonstrate that genetic immunization by in vivo EP is more efficient than previous procedures, and that it is useful for delineating the pathophysiology of Graves' disease.

C-phycocyanin extraction assisted by pulsed electric field from Artrosphira platensis.

PubMed

Martínez, Juan Manuel; Luengo, Elisa; Saldaña, Guillermo; Álvarez, Ignacio; Raso, Javier

2017-09-01

This paper assesses the application of pulsed electric fields (PEF) to the fresh biomass of Artrhospira platensis in order to enhance the extraction of C-phycocyanin into aqueous media. Electroporation of A. platensis depended on both electric field strength and treatment duration. The minimum electric field intensity for detecting C-phycocyanin in the extraction medium was 15kV/cm after the application of a treatment time 150μs (50 pulses of 3μs). However higher electric field strength were required when shorter treatment times were applied. Response surface methodology was used in order to investigate the influence of electric field strength (15-25kV/cm), treatment time (60-150μs), and temperature of application of PEF (10-40°C) on C-phycocyanin extraction yield (PEY). The increment of the temperature PEF treatment reduced the electric field strength and the treatment time required to obtain a given PEY and, consequently decreased the total specific energy delivered by the treatment. For example, the increment of temperature from 10°C to 40°C permitted to reduce the electric field strength required to extract 100mg/g d w of C-phycocyanin from 25 to 18kV/cm, and the specific energy input from 106.7 to 67.5kJ/Kg. Results obtained in this investigation demonstrated PEF's potential for selectively extraction C-phycocyanin from fresh A. platensis biomass. The purity of the C-phycocyanin extract obtained from the electroporated cells was higher than that obtained using other techniques based on the cell complete destruction. Copyright © 2016 Elsevier Ltd. All rights reserved.

Recombineering: A Homologous Recombination-Based Method of Genetic Engineering

PubMed Central

Sharan, Shyam K.; Thomason, Lynn C.; Kuznetsov, Sergey G.; Court, Donald L.

2009-01-01

Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in E. coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraints being imposed by restriction enzyme site location. Bacteriophage homologous recombination proteins catalyze these recombineering reactions using double- and single-strand linear DNA substrates, so-called targeting constructs, introduced by electroporation. Gene knockouts, deletions and point mutations are readily made, gene tags can be inserted, and regions of bacterial artificial chromosomes (BACs) or the E. coli genome can be subcloned by gene retrieval using recombineering. Most of these constructs can be made within about a week's time. PMID:19180090

Gene knockout and overexpression analysis revealed the role of N-acetylmuramidase in autolysis of Lactobacillus delbrueckii subsp. bulgaricus ljj-6.

PubMed

Pang, Xiao-Yang; Cui, Wen-Ming; Liu, Lu; Zhang, Shu-Wen; Lv, Jia-Ping

2014-01-01

Autolysis of lactic acid bacteria (LAB) plays a vital role in dairy processing. During cheese making, autolysis of LAB affects cheese flavor development through release of intracellular enzymes and restricts the proliferation of cells in yogurt fermentation and probiotics production. In order to explore the mechanism of autolysis, the gene for the autolytic enzymes of L. bulgaricus, N-acetylmuramidase (mur), was cloned and sequenced (GenBank accession number: KF157911). Mur gene overexpression and gene knockout vectors were constructed based on pMG76e and pUC19 vectors. Recombinant plasmids were transformed into L. bulgaricus ljj-6 by electroporation, then three engineered strains with pMG76e-mur vector and fifteen engineered strains with pUC19-mur::EryBII were screened. The autolysis of the mur knockout strain was significantly lower and autolysis of the mur overexpressed strain was significantly higher compared with that of the wild type strain ljj-6. This result suggested that the mur gene played an important role in autolysis of L. bulgaricus. On the other hand, autolytic activity in a low degree was still observed in the mur knockout strain, which implied that other enzymes but autolysin encoded by mur were also involved in autolysis of L. bulgaricus.

Method and apparatus for making articles from particle based materials

DOEpatents

Moorhead, Arthur J.; Menchhofer, Paul A.

1995-01-01

A method and apparatus for the production of articles made of a particle-based material; e.g., ceramics and sintered metals. In accordance with the invention, a thermally settable slurry containing a relatively high concentration of the particles is conveyed through an elongate flow area having a desired cross-sectional configuration. The slurry is heated as it is advanced through the flow area causing the slurry to set or harden in a shape which conforms to the cross-sectional configuration of the flow area. The material discharges from the flow area as a self-supporting solid of near net final dimensions. The article may then be sintered to consolidate the particles and provide a high density product.

Transdermal drug delivery

PubMed Central

Prausnitz, Mark R.; Langer, Robert

2009-01-01

Transdermal drug delivery has made an important contribution to medical practice, but has yet to fully achieve its potential as an alternative to oral delivery and hypodermic injections. First-generation transdermal delivery systems have continued their steady increase in clinical use for delivery of small, lipophilic, low-dose drugs. Second-generation delivery systems using chemical enhancers, non-cavitational ultrasound and iontophoresis have also resulted in clinical products; the ability of iontophoresis to control delivery rates in real time provides added functionality. Third-generation delivery systems target their effects to skin’s barrier layer of stratum corneum using microneedles, thermal ablation, microdermabrasion, electroporation and cavitational ultrasound. Microneedles and thermal ablation are currently progressing through clinical trials for delivery of macromolecules and vaccines, such as insulin, parathyroid hormone and influenza vaccine. Using these novel second- and third-generation enhancement strategies, transdermal delivery is poised to significantly increase impact on medicine. PMID:18997767

Time-Dependent Impact of Irreversible Electroporation on Pancreas, Liver, Blood Vessels and Nerves: A Systematic Review of Experimental Studies

PubMed Central

Agnass, P.; Crezee, J.; Dijk, F.; Verheij, J.; van Gulik, T. M.; Meijerink, M. R.; Vroomen, L. G.; van Lienden, K. P.; Besselink, M. G.

2016-01-01

Introduction Irreversible electroporation (IRE) is a novel ablation technique in the treatment of unresectable cancer. The non-thermal mechanism is thought to cause mostly apoptosis compared to necrosis in thermal techniques. Both in experimental and clinical studies, a waiting time between ablation and tissue or imaging analysis to allow for cell death through apoptosis, is often reported. However, the dynamics of the IRE effect over time remain unknown. Therefore, this study aims to summarize these effects in relation to the time between treatment and evaluation. Methods A systematic search was performed in Pubmed, Embase and the Cochrane Library for original articles using IRE on pancreas, liver or surrounding structures in animal or human studies. Data on pathology and time between IRE and evaluation were extracted. Results Of 2602 screened studies, 36 could be included, regarding IRE in liver (n = 24), pancreas (n = 4), blood vessels (n = 4) and nerves (n = 4) in over 440 animals (pig, rat, goat and rabbit). No eligible human studies were found. In liver and pancreas, the first signs of apoptosis and haemorrhage were observed 1–2 hours after treatment, and remained visible until 24 hours in liver and 7 days in pancreas after which the damaged tissue was replaced by fibrosis. In solitary blood vessels, the tunica media, intima and lumen remained unchanged for 24 hours. After 7 days, inflammation, fibrosis and loss of smooth muscle cells were demonstrated, which persisted until 35 days. In nerves, the median time until demonstrable histological changes was 7 days. Conclusions Tissue damage after IRE is a dynamic process with remarkable time differences between tissues in animals. Whereas pancreas and liver showed the first damages after 1–2 hours, this took 24 hours in blood vessels and 7 days in nerves. PMID:27870918

Fiber-optic system for checking the acoustical parameters of gas-turbine engine flow-through passages

NASA Astrophysics Data System (ADS)

Vinogradov, Vasiliy Y.; Morozov, Oleg G.; Nureev, Ilnur I.; Kuznetzov, Artem A.

2015-03-01

In this paper we consider the integrated approach to development of the aero-acoustical methods for diagnostics of aircraft gas-turbine engine flow-through passages by using as the base the passive fiber-optic and location technologies.

Study of compressible flow through a rectangular-to-semiannular transition duct

NASA Technical Reports Server (NTRS)

Foster, Jeffry; Okiishi, Theodore H.; Wendt, Bruce J.; Reichert, Bruce A.

1995-01-01

Detailed flow field measurements are presented for compressible flow through a diffusing rectangular-to-semiannular transition duct. Comparisons are made with published computational results for flow through the duct. Three-dimensional velocity vectors and total pressures were measured at the exit plane of the diffuser model. The inlet flow was also measured. These measurements are made using calibrated five-hole probes. Surface oil flow visualization and surface static pressure data were also taken. The study was conducted with an inlet Mach number of 0.786. The diffuser Reynolds based on the inlet centerline velocity and the exit diameter of the diffuser was 3,200,000. Comparison of the measured data with previously published computational results are made. Data demonstrating the ability of vortex generators to reduce flow separation and circumferential distortion is also presented.

Agricultural and Food Processing Applications of Pulsed Power Technology

NASA Astrophysics Data System (ADS)

Takaki, Koichi; Ihara, Satoshi

Recent progress of agricultural and food processing applications of pulsed power is described in this paper. Repetitively operated compact pulsed power generators with a moderate peak power have been developed for the agricultural and the food processing applications. These applications are mainly based on biological effects and can be categorized as decontamination of air and liquid, germination promotion, inhabitation of saprophytes growth, extraction of juice from fruits and vegetables, and fertilization of liquid medium, etc. Types of pulsed power that have biological effects are caused with gas discharges, water discharges, and electromagnetic fields. The discharges yield free radicals, UV radiation, intense electric field, and shock waves. Biologically based applications of pulsed power are performed by selecting the type that gives the target objects the adequate result from among these agents or byproducts. For instance, intense electric fields form pores on the cell membrane, which is called electroporation, or influence the nuclei.

Reduction of Werner Syndrome Protein Enhances G:C → A:T Transition by O6-Methylguanine in Human Cells.

PubMed

Suzuki, Tetsuya; Kuramoto, Yoshie; Kamiya, Hiroyuki

2018-05-21

O 6 -Methylguanine ( O 6 -MeG) is a damaged base produced by methylating reagents. The Werner syndrome protein (WRN) is a cancer-related human DNA helicase. The effects of WRN reduction on O 6 -MeG-caused mutagenesis were assessed by an siRNA-mediated knockdown in human U2OS cells, using a shuttle plasmid with a single O 6 -MeG base in the supF gene. The plasmid DNA was replicated in the cells, isolated, and electroporated into an Escherichia coli indicator strain. The lowered amount of WRN increased the frequency of mutations induced by O 6 -MeG, mainly G:C → A:T substitution. The increased mutation rate suggested that the cancer-related WRN suppresses the G:C → A:T substitution by O 6 -MeG in human cells.

A microfluidic fuel cell with flow-through porous electrodes.

PubMed

Kjeang, Erik; Michel, Raphaelle; Harrington, David A; Djilali, Ned; Sinton, David

2008-03-26

A microfluidic fuel cell architecture incorporating flow-through porous electrodes is demonstrated. The design is based on cross-flow of aqueous vanadium redox species through the electrodes into an orthogonally arranged co-laminar exit channel, where the waste solutions provide ionic charge transfer in a membraneless configuration. This flow-through architecture enables improved utilization of the three-dimensional active area inside the porous electrodes and provides enhanced rates of convective/diffusive transport without increasing the parasitic loss required to drive the flow. Prototype fuel cells are fabricated by rapid prototyping with total material cost estimated at 2 USD/unit. Improved performance as compared to previous microfluidic fuel cells is demonstrated, including power densities at room temperature up to 131 mW cm-2. In addition, high overall energy conversion efficiency is obtained through a combination of relatively high levels of fuel utilization and cell voltage. When operated at 1 microL min-1 flow rate, the fuel cell produced 20 mW cm-2 at 0.8 V combined with an active fuel utilization of 94%. Finally, we demonstrate in situ fuel and oxidant regeneration by running the flow-through architecture fuel cell in reverse.

Review Of Applied Mathematical Models For Describing The Behaviour Of Aqueous Humor In Eye Structures

NASA Astrophysics Data System (ADS)

Dzierka, M.; Jurczak, P.

2015-12-01

In the paper, currently used methods for modeling the flow of the aqueous humor through eye structures are presented. Then a computational model based on rheological models of Newtonian and non-Newtonian fluids is proposed. The proposed model may be used for modeling the flow of the aqueous humor through the trabecular meshwork. The trabecular meshwork is modeled as an array of rectilinear parallel capillary tubes. The flow of Newtonian and non-Newtonian fluids is considered. As a results of discussion mathematical equations of permeability of porous media and velocity of fluid flow through porous media have been received.

PageRank versatility analysis of multilayer modality-based network for exploring the evolution of oil-water slug flow.

PubMed

Gao, Zhong-Ke; Dang, Wei-Dong; Li, Shan; Yang, Yu-Xuan; Wang, Hong-Tao; Sheng, Jing-Ran; Wang, Xiao-Fan

2017-07-14

Numerous irregular flow structures exist in the complicated multiphase flow and result in lots of disparate spatial dynamical flow behaviors. The vertical oil-water slug flow continually attracts plenty of research interests on account of its significant importance. Based on the spatial transient flow information acquired through our designed double-layer distributed-sector conductance sensor, we construct multilayer modality-based network to encode the intricate spatial flow behavior. Particularly, we calculate the PageRank versatility and multilayer weighted clustering coefficient to quantitatively explore the inferred multilayer modality-based networks. Our analysis allows characterizing the complicated evolution of oil-water slug flow, from the opening formation of oil slugs, to the succedent inter-collision and coalescence among oil slugs, and then to the dispersed oil bubbles. These properties render our developed method particularly powerful for mining the essential flow features from the multilayer sensor measurements.

Oxygen-Mass-Flow Calibration Cell

NASA Technical Reports Server (NTRS)

Martin, Robert E.

1996-01-01

Proposed calibration standard for mass flow rate of oxygen based on conduction of oxygen ions through solid electrolyte membrane made of zirconia and heated to temperature of 1,000 degrees C. Flow of oxygen ions proportional to applied electric current. Unaffected by variations in temperature and pressure, and requires no measurement of volume. Calibration cell based on concept used to calibrate variety of medical and scientific instruments required to operate with precise rates of flow of oxygen.

Numerical models of jet disruption in cluster cooling flows

NASA Technical Reports Server (NTRS)

Loken, Chris; Burns, Jack O.; Roettiger, Kurt; Norman, Mike

1993-01-01

We present a coherent picture for the formation of the observed diverse radio morphological structures in dominant cluster galaxies based on the jet Mach number. Realistic, supersonic, steady-state cooling flow atmospheres are evolved numerically and then used as the ambient medium through which jets of various properties are propagated. Low Mach number jets effectively stagnate due to the ram pressure of the cooling flow atmosphere while medium Mach number jets become unstable and disrupt in the cooling flow to form amorphous structures. High Mach number jets manage to avoid disruption and are able to propagate through the cooling flow.

Effect of heat transfer on rotating electroosmotic flow through a micro-vessel: haemodynamical applications

NASA Astrophysics Data System (ADS)

Sinha, A.; Mondal, A.; Shit, G. C.; Kundu, P. K.

2016-08-01

This paper theoretically analyzes the heat transfer characteristics associated with electroosmotic flow of blood through a micro-vessel having permeable walls. The analysis is based on the Debye-Hückel approximation for charge distributions and the Navier-Stokes equations are assumed to represent the flow field in a rotating system. The velocity slip condition at the vessel walls is taken into account. The essential features of the rotating electroosmotic flow of blood and associated heat transfer characteristics through a micro-vessel are clearly highlighted by the variation in the non-dimensional flow velocity, volumetric flow rate and non-dimensional temperature profiles. Moreover, the effect of Joule heating parameter and Prandtl number on the thermal transport characteristics are discussed thoroughly. The study reveals that the flow of blood is appreciably influenced by the elctroosmotic parameter as well as rotating Reynolds number.

Research on virtual network load balancing based on OpenFlow

NASA Astrophysics Data System (ADS)

Peng, Rong; Ding, Lei

2017-08-01

The Network based on OpenFlow technology separate the control module and data forwarding module. Global deployment of load balancing strategy through network view of control plane is fast and of high efficiency. This paper proposes a Weighted Round-Robin Scheduling algorithm for virtual network and a load balancing plan for server load based on OpenFlow. Load of service nodes and load balancing tasks distribution algorithm will be taken into account.

Method and apparatus for making articles from particle based materials

DOEpatents

Moorhead, A.J.; Menchhofer, P.A.

1995-12-19

A method and apparatus are disclosed for the production of articles made of a particle-based material; e.g., ceramics and sintered metals. In accordance with the invention, a thermally settable slurry containing a relatively high concentration of the particles is conveyed through an elongate flow area having a desired cross-sectional configuration. The slurry is heated as it is advanced through the flow area causing the slurry to set or harden in a shape which conforms to the cross-sectional configuration of the flow area. The material discharges from the flow area as a self-supporting solid of near net final dimensions. The article may then be sintered to consolidate the particles and provide a high density product. 10 figs.

Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, Masahito; Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571; Umeyama, Kazuhiro

2010-11-05

Research highlights: {yields} EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. {yields} ZFNs induced targeted mutations in porcine primary cultured cells. {yields} Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor themore » exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.« less

Time-of-flight dependency on transducer separation distance in a reflective-path guided-wave ultrasonic flow meter at zero flow conditions.

PubMed

Aanes, Magne; Kippersund, Remi Andre; Lohne, Kjetil Daae; Frøysa, Kjell-Eivind; Lunde, Per

2017-08-01

Transit-time flow meters based on guided ultrasonic wave propagation in the pipe spool have several advantages compared to traditional inline ultrasonic flow metering. The extended interrogation field, obtained by continuous leakage from guided waves traveling in the pipe wall, increases robustness toward entrained particles or gas in the flow. In reflective-path guided-wave ultrasonic flow meters (GW-UFMs), the flow equations are derived from signals propagating solely in the pipe wall and from signals passing twice through the fluid. In addition to the time-of-flight (TOF) through the fluid, the fluid path experiences an additional time delay upon reflection at the opposite pipe wall due to specular and non-specular reflections. The present work investigates the influence of these reflections on the TOF in a reflective-path GW-UFM as a function of transducer separation distance at zero flow conditions. Two models are used to describe the signal propagation through the system: (i) a transient full-wave finite element model, and (ii) a combined plane-wave and ray-tracing model. The study shows that a range-dependent time delay is associated with the reflection of the fluid path, introducing transmitter-receiver distance dependence. Based on these results, the applicability of the flow equations derived using model (ii) is discussed.

DNA Immunization

PubMed Central

Wang, Shixia; Lu, Shan

2013-01-01

DNA immunization was discovered in early 1990s and its use has been expanded from vaccine studies to a broader range of biomedical research, such as the generation of high quality polyclonal and monoclonal antibodies as research reagents. In this unit, three common DNA immunization methods are described: needle injection, electroporation and gene gun. In addition, several common considerations related to DNA immunization are discussed. PMID:24510291

DNA electrotransfer into the skin using a combination of one high- and one low-voltage pulse.

PubMed

Pavselj, N; Préat, V

2005-09-02

Electroporation is an effective alternative to viral methods to significantly improve DNA transfection after intradermal and topical delivery. The aim of the study was to check whether a combination of a short high-voltage pulse (HV) to permeabilize the skin cells and a long low-voltage pulse (LV) to transfer DNA by electrophoresis was more efficient to enhance DNA expression than conventional repeated HV or LV pulses alone after intradermal injection of DNA plasmid. GFP and luciferase expressions in the skin were enhanced by HV+LV protocol as compared to HV or LV pulses alone. The expression lasted for up to 10 days. Consistently, HV+LV protocol induced a higher Th2 immune response against ovalbumin than HV or LV pulses. Standard methods were used to assess the effect of electric pulses on skin: the application of a combination of HV and LV pulses on rat skin fold delivered by plate electrodes was well tolerated. These data demonstrate that a combination of one HV (700 to 1000 V/cm; 100 micros) followed by one LV (140 to 200 V/cm; 400 ms) is an efficient electroporation protocol to enhance DNA expression in the skin.

Electroporation of the Testis

NASA Astrophysics Data System (ADS)

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.

Mechanisms of antimelanoma effect of oat β-glucan supported by electroporation.

PubMed

Choromanska, Anna; Lubinska, Sandra; Szewczyk, Anna; Saczko, Jolanta; Kulbacka, Julita

2018-06-06

There are still not specified mechanisms how beta-glucan molecules are transported into cells. Supposing, beta-glucan toxicity against tumor cells may be related to the overexpression of the transporter responsible for the transport of glucose molecules in the cells. In this case, glucans - polymers composed of glucose units are much more up-taken by tumor than normal cells. Increased GLUT1 (Glucose Transporter Type 1) expression has been demonstrated earlier in malignant melanomas. GLUT1 expression promotes glucose uptake and cell growth in that cells. Also, in human melanoma tissues a significant correlation between GLUT1 expression and mitotic activity was found. The aim of the study was to verify if oat β-glucan (OβG) is delivered into cells by GLUT-1 membrane protein. To check it out we blocked GLUT1 transporters by an inhibitor WZB117 and then we investigated cells viability with and without reversible electroporation (EP). The obtained results bring us to elucidate the mechanism of transport of the OβG into the cells is GLUT-1 dependent and moreover can be supported by EP method. Copyright © 2018. Published by Elsevier B.V.

Irreversible electroporation and the pancreas: What we know and where we are going?

PubMed

Young, Shamar J

2015-08-27

Pancreatic adenocarcinoma continues to have a poor prognosis with 1 and 5 years survival rates of 27% and 6% respectively. The gold standard of treatment is resection, however, only approximately 10% of patients present with resectable disease. Approximately 40% of patients present with disease that is too locally advanced to resect. There is great interest in improving outcomes in this patient population and ablation techniques have been investigated as a potential solution. Unfortunately early investigations into thermal ablation techniques, particularly radiofrequency ablation, resulted in unacceptably high morbidity rates. Irreversible electroporation (IRE) has been introduced and is promising as it does not rely on thermal energy and has shown an ability to leave structural cells such as blood vessels and bile ducts intact during animal studies. IRE also does not suffer from heat sink effect, a concern given the large number of blood vessels surrounding the pancreas. IRE showed significant promise during preclinical animal trials and as such has moved on to clinical testing. There are as of yet only a few studies which look at the applications of IRE within humans in the setting of pancreatic adenocarcinoma. This paper reviews the basic principles, techniques, and current clinical data available on IRE.

The hominoid-specific gene TBC1D3 promotes generation of basal neural progenitors and induces cortical folding in mice

PubMed Central

Ju, Xiang-Chun; Hou, Qiong-Qiong; Sheng, Ai-Li; Wu, Kong-Yan; Zhou, Yang; Jin, Ying; Wen, Tieqiao; Yang, Zhengang; Wang, Xiaoqun; Luo, Zhen-Ge

2016-01-01

Cortical expansion and folding are often linked to the evolution of higher intelligence, but molecular and cellular mechanisms underlying cortical folding remain poorly understood. The hominoid-specific gene TBC1D3 undergoes segmental duplications during hominoid evolution, but its role in brain development has not been explored. Here, we found that expression of TBC1D3 in ventricular cortical progenitors of mice via in utero electroporation caused delamination of ventricular radial glia cells (vRGs) and promoted generation of self-renewing basal progenitors with typical morphology of outer radial glia (oRG), which are most abundant in primates. Furthermore, down-regulation of TBC1D3 in cultured human brain slices decreased generation of oRGs. Interestingly, localized oRG proliferation resulting from either in utero electroporation or transgenic expression of TBC1D3, was often found to underlie cortical regions exhibiting folding. Thus, we have identified a hominoid gene that is required for oRG generation in regulating the cortical expansion and folding. DOI: http://dx.doi.org/10.7554/eLife.18197.001 PMID:27504805

Electroporation-assisted discrimination of normal, benign and cancerous human gastric tissues by OCT and diffuse reflectance spectra images

NASA Astrophysics Data System (ADS)

Li, Caiyun; Wei, Huajiang; Zhao, Yanping; Wu, Guoyong; Gu, Huaimin; Guo, Zhouyi; Yang, Hongqin; He, Yonghong; Xie, Shusen

2018-07-01

The purpose of this study is to illustrate experimentally the optical coherence tomography (OCT) signal slope and diffuse reflectance (DR) spectra of 30% and 80% glycerol combined with electroporation (EP) diffusion in normal, benign and cancerous human gastric tissues in vitro. The results of OCT showed that the permeability coefficients of 80% and 30% glycerol (both with and without EP) have the following trend: human cancerous gastric tissue  >  human benign gastric tissue  >  human normal gastric tissue under the same conditions. The permeability coefficient of the 30% glycerol group is larger than that of the 80% glycerol group under the same circumstances; the permeability coefficient of glycerol combined with the EP group is larger than that without the EP group under the same conditions. The permeability coefficient and the reduction of the DR spectra have perfect linear correlation (R2  =  0.9745). The research results suggest that OCT and the DR spectra combined with an optical clearing agent (glycerol) and the EP method can potentially become a powerful tool for the early diagnosis and monitoring of human gastric cancer.

Factors affecting the efficient transformation of Colletotrichum species

USGS Publications Warehouse

Redman, Regina S.; Rodriguez, Rusty J.

1994-01-01

Factors affecting the efficient transformation of Colletotrichum species. Experimental Mycology, 18, 230-246. Twelve isolates representing four species of Colletotrichum were transformed either by enhanced protoplast, restriction enzyme-mediated integration (REMI), or electroporation-mediated protocols. The enhanced protoplast transformation protocol resulted in 100- and 50-fold increases in the transformation efficiencies of Colletotrichum lindemuthianum and C. magna , respectively. REMI transformation involved the use of Hin dIII and vector DNA linearized with HindIII to increase the number of integration events and potential gene disruptions in the fungal genome. Combining the enhanced protoplast and the REMI protocols resulted in a 22-fold increase in the number of hygromycin/nystatin-resistant mutants in C. lindemuthianum . Electroporation-mediated transformation was performed on mycelial fragments and spores of four Colletotrichum species, resulting in efficiencies of up to 1000 transformants/μg DNA. The pHA1.3 vector which confers hygromycin resistance contains telomeric sequences from Fusarium oxysporum , transforms by autonomous replication and genomic integration, and was essential for elevated transformation efficiencies of 100 to 10,000 transformants/μg DNA. Modifications of pHA1.3 occurred during bacterial amplification and post fungal transformation resulting in plasmids capable of significantly elevated transformation efficiencies in C. lindemuthianum.

Effects of high voltage nanosecond electric pulses on eukaryotic cells (in vitro): A systematic review.

PubMed

Batista Napotnik, Tina; Reberšek, Matej; Vernier, P Thomas; Mali, Barbara; Miklavčič, Damijan

2016-08-01

For this systematic review, 203 published reports on effects of electroporation using nanosecond high-voltage electric pulses (nsEP) on eukaryotic cells (human, animal, plant) in vitro were analyzed. A field synopsis summarizes current published data in the field with respect to publication year, cell types, exposure configuration, and pulse duration. Published data were analyzed for effects observed in eight main target areas (plasma membrane, intracellular, apoptosis, calcium level and distribution, survival, nucleus, mitochondria, stress) and an additional 107 detailed outcomes. We statistically analyzed effects of nsEP with respect to three pulse duration groups: A: 1-10ns, B: 11-100ns and C: 101-999ns. The analysis confirmed that the plasma membrane is more affected with longer pulses than with short pulses, seen best in uptake of dye molecules after applying single pulses. Additionally, we have reviewed measurements of nsEP and evaluations of the electric fields to which cells were exposed in these reports, and we provide recommendations for assessing nanosecond pulsed electric field effects in electroporation studies. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Magnetoelectricity in Multi-Scale Composites and Application in Nanorobotics for Live Cell Manipulation

NASA Astrophysics Data System (ADS)

Betal, Soutik

In this research biomedical and sensor applications of magnetoelectric effect have been broadly explored using magnetoelectric composites. Firstly NiFe2O4/Pb(Zr0.52Ti0.48)O 3/NiFe2O4 layered bulk composite have been studied to achieve high magnetoelectric coefficient for their applications in brain magnetic field detection at room temperature. Magnetic sensors like SQUID (superconducting quantum interference device) nowadays are able to detect pico-Tesla magnetic fields produced outside the brain by the neuronal currents which can be used for diagnostic application, but due to heavy liquid helium cooling and insulation requirements, the technique become quite inefficient in gaining high resolution measurement. At room temperature layered ME samples exhibit high magnetoelectric response in mV/cm.Oe range and hence can transform very low magnetic field into electric signal which can be measured even in femtovolts. Moreover temperature and a.c. frequency dependent studies were done to extensively characterize the layered ME sample for sensor application. Secondly core-shell magnetoelectric nanoparticles (CSMEN) have been fabricated, characterized and their interaction with biological cell in presence of a.c. and d.c. field have been thoroughly analyzed. A magnetically controlled elastically driven electroporation phenomenon, or Magneto-Elasto- Electroporation (MEEP), is discovered while studying interactions between core-shell magneto-electric nanoparticles (CSMEN) and biological cells in the presence of an AC magnetic field. In this research MEEP effect was observed via a series of in-vitro experiments using core (CoFe2O4)-shell (BaTiO3 ) structured magnetoelectric nanoparticles and human epithelial cells (HEP2). Cell electroporation phenomenon and its correlation with the magnetic field modulated CSMEN have been elaborately studied. Potential of CSMEN for application in targeted single cell electroporation have been confirmed by analysing crystallographic phases, multiferroic properties of the fabricated CSMEN , influences of DC and AC magnetic field on the CSMEN and cytotoxicity tests. We also report the mathematical formalism to quantitatively describe the phenomena. The reported findings provide the basis of the underlying MEEP mechanism and demonstrate the utility of CSMEN as electric pulse generating nano-probe in cell electroporation experiments for the potential application towards accurate and efficient targeted cell permeation as well as drug delivery. Thirdly, experiments of fabricated magnetoelectric nanocomposites with biological cells in controlled boundary condition under fluctuating and biased magnetic field excitation revealed the smart nanorobotics characteristics of the nanostructure to achieve remote controlled dynamically targeted live cell manipulation. A remotely controlled dynamic process of manipulating targeted biological live cells using fabricated core-shell magnetoelectric nanocomposites have been fabricated, which comprises of single crystalline ferromagnetic cores (CoFe2O4) coated with crystalline ferroelectric thin film shells (BaTiO3). These nanocomposites are demonstrated as a unique family of inorganic magnetoelectric nanorobots (MENRs), controlled remotely by applied a.c. or d.c. magnetic fields, to perform cell targeting, permeation, patterning and transport. MENRs performs these functions via localized electric periodic pulse generation, local electric-field sensing, or thrust generation and acts as a unique tool for remotely controlled dynamically targeted cellular manipulation. Under a.c. magnetic field excitation (50 Oe, 60 Hz), the MENR acts as a localized periodic electric pulse generator and can permeate a series of misaligned cells, while aligning/patterning them to an equipotential mono-array. Under a.c. magnetic field (40 Oe, 30 Hz) excitation, MENRs can be dynamically driven to a targeted cell, avoiding untargeted cells in the path, irrespective of cell density. D.C. magnetic field (-50 Oe) excitation causes the MENRs to act as thrust generator and exerts motion in a group of cells. Visualization of magnetoelectricity at nanoscale and its application in dynamically targeted live cell manipulation have been presented in this research.

An Alternating Current Electroosmotic Pump Based on Conical Nanopore Membranes.

PubMed

Wu, Xiaojian; Ramiah Rajasekaran, Pradeep; Martin, Charles R

2016-04-26

Electroosmotic flow (EOF) is used to pump solutions through microfluidic devices and capillary electrophoresis columns. We describe here an EOF pump based on membrane EOF rectification, an electrokinetic phenomenon we recently described. EOF rectification requires membranes with asymmetrically shaped pores, and conical pores in a polymeric membrane were used here. We show here that solution flow through the membrane can be achieved by applying a symmetrical sinusoidal voltage waveform across the membrane. This is possible because the alternating current (AC) carried by ions through the pore is rectified, and we previously showed that rectified currents yield EOF rectification. We have investigated the effect of both the magnitude and frequency of the voltage waveform on flow rate through the membrane, and we have measured the maximum operating pressure. Finally, we show that operating in AC mode offers potential advantages relative to conventional DC-mode EOF pumps.

Universal chemiluminescence flow-through device based on directed self-assembly of solid-state organic chromophores on layered double hydroxide matrix.

PubMed

Wang, Zhihua; Teng, Xu; Lu, Chao

2013-02-19

In this work, a universal chemiluminescence (CL) flow-through device suitable for various CL resonance energy transfer (CRET) systems has been successfully fabricated. Highly efficient CRET in solid-state photoactive organic molecules can be achieved by assembling them on the surface of layered double hydroxides (LDHs). We attribute these observations to the suppression of the intermolecular π-π stacking interactions among aromatic rings and the improvement of molecular orientation and planarity in the LDH matrix, enabling a remarkable increase in fluorescence lifetime and quantum yield of organic molecules. Under optimal conditions, using peroxynitrous acid-fluorescein dianion (FLUD) as a model CRET system, trace FLUD (10 μM) was assembled on the surface of LDHs. Peroxynitrous acid/nitrite could be assayed in the range of 1.0-500 μM, and the detection limit for peroxynitrous acid/nitrite (S/N = 3) was 0.6 μM. This CL flow-through device exhibited operational stability, high reproducibility, and long lifetime. While LDHs were immobilized in a flow-through device in the absence of FLUD, the detection limit for peroxynitrous acid/nitrite was 100 μM. On the other hand, FLUD at the same concentration can not enhance the CL intensity of peroxynitrous acid system. This fabricated CL flow-through column has been successfully applied to determine nitrite in sausage samples with recoveries of 98-102%. These satisfactory results demonstrated that our studies pave a novel way toward flow-through column-based CRET using solid-state organic molecules as acceptors for signal amplification.

Redesigning flow injection after 40 years of development: Flow programming.

PubMed

Ruzicka, Jaromir Jarda

2018-01-01

Automation of reagent based assays, by means of Flow Injection (FI), is based on sample processing, in which a sample flows continuously towards and through a detector for quantification of the target analyte. The Achilles heel of this methodology, the legacy of Auto Analyzer®, is continuous reagent consumption, and continuous generation of chemical waste. However, flow programming, assisted by recent advances in precise pumping, combined with the lab-on-valve technique, allows the FI manifold to be designed around a single confluence point through which sample and reagents are sequentially directed by means of a series of flow reversals. This approach results in sample/reagent mixing analogous to the traditional FI, reduces sample and reagent consumption, and uses the stop flow technique for enhancement of the yield of chemical reactions. The feasibility of programmable Flow Injection (pFI) is documented by example of commonly used spectrophotometric assays of, phosphate, nitrate, nitrite and glucose. Experimental details and additional information are available in online tutorial http://www.flowinjectiontutorial.com/. Copyright © 2017 Elsevier B.V. All rights reserved.

Through-Flow Calculations in Axial Turbomachinery

DTIC Science & Technology

1976-10-01

coilditions should be next on the agenda. Authors’ response: I think the process is essentially iterative between SI and S2 solutions. If SI surfaces...secondary flows in high Mach number situations. Concerning Gelder’s approach, i think that your remark is rather optimistic. We use a method based on...my remarks on Gelder’s work were based on calculations made by Gelder himself. One or two other people have managed to get the calculation through

Urethral anatomy and semen flow during ejaculation

NASA Astrophysics Data System (ADS)

Kelly, Diane

2016-11-01

Ejaculation is critical for reproductive success in many animals, but little is known about its hydrodynamics. In mammals, ejaculation pushes semen along the length of the penis through the urethra. Although the urethra also carries urine during micturition, the flow dynamics of micturition and ejaculation differ: semen is more viscous than urine, and the pressure that drives its flow is derived primarily from the rhythmic contractions of muscles at the base of the penis, which produce pulsatile rather than steady flow. In contrast, Johnston et al. (2014) describe a steady flow of semen through the crocodilian urethral groove during ejaculation. Anatomical differences of tissues associated with mammalian and crocodilian urethral structures may underlie these differences in flow behavior.

Accounting For Compressibility In Viscous Flow In Pipes

NASA Technical Reports Server (NTRS)

Steinle, Frank W.; Gee, Ken; Murthy, Sreedhara V.

1991-01-01

Method developed to account for effects of compressibility in viscous flows through long, circular pipes of uniform diameter. Based on approximation of variations in density and velocity across pipe cross section by profile equations developed for boundary-layer flow between flat plates.

Peak-flow frequency analyses and results based on data through water year 2011 for selected streamflow-gaging stations in or near Montana: Chapter C in Montana StreamStats

USGS Publications Warehouse

Sando, Steven K.; McCarthy, Peter M.; Dutton, DeAnn M.

2016-04-05

Chapter C of this Scientific Investigations Report documents results from a study by the U.S. Geological Survey, in cooperation with the Montana Department of Transportation and the Montana Department of Natural Resources, to provide an update of statewide peak-flow frequency analyses and results for Montana. The purpose of this report chapter is to present peak-flow frequency analyses and results for 725 streamflow-gaging stations in or near Montana based on data through water year 2011. The 725 streamflow-gaging stations included in this study represent nearly all streamflowgaging stations in Montana (plus some from adjacent states or Canadian Provinces) that have at least 10 years of peak-flow records through water year 2011. For 29 of the 725 streamflow-gaging stations, peak-flow frequency analyses and results are reported for both unregulated and regulated conditions. Thus, peak-flow frequency analyses and results are reported for a total of 754 analyses. Estimates of peak-flow magnitudes for 66.7-, 50-, 42.9-, 20-, 10-, 4-, 2-, 1-, 0.5-, and 0.2-percent annual exceedance probabilities are reported. These annual exceedance probabilities correspond to 1.5-, 2-, 2.33-, 5-, 10-, 25-, 50-, 100-, 200-, and 500-year recurrence intervals.

An important discovery on combination of irreversible electroporation and allogeneic natural killer cell immunotherapy for unresectable pancreatic cancer

PubMed Central

Liang, Shuzhen; Wang, Xiaohua; Liang, Yinqing; Zhang, Mingjie; Chen, Jibing; Niu, Lizhi; Xu, Kecheng

2017-01-01

Purpose To study the safety and clinical efficacy on combination of irreversible electroporation and allogeneic natural killer cell therapy for treating Stage III/IV pancreatic cancer, evaluating median progression free survival (PFS), and overall survival (OS). Results Adverse events of all patients were limited to grades 1 and 2, including local (mainly tussis 13.4%, nausea and emesis 7.1%, pain of puncture point 29.6% and duodenum and gastric retention 4.3%) and systemic (mainly fatigue 22.3%, fever 31.6%, and transient reduction of intraoperative blood pressure 25.1% and white cell count reduction 18.3%) reactions, fever was the most frequent. The serum amylase level at 24 h and 7 d after IRE was not significantly changed compared to those before IRE (P > 0.05). CA19–9 value was lower in IRE-NK group than in IRE at 1 month after treatment (P < 0.05). After a median follow-up of 7.4 months (3.6–11.2 months): in stage III group, median PFS was higher in IRE-NK group (9.3 months) than in IRE group (8.1 months, P = 0.0465), median OS was higher in IRE-NK (13.2 months) than in IRE (11.4 months, P = 0.0411), and median PFS was higher in who received multiple NK than single NK (9.8 months vs.8.1 months, P = 0.0423, respectively), median OS who received multiple NK was higher than single NK (13.9 months vs.12.3 months, P = 0.0524, respectively), the RR in IRE-NK (63.2%) was higher than in IRE (50.0%, P < 0.05); in stage IV group, median OS was higher in IRE-NK (9.8 months) than in IRE (8.7 months, P = 0.0397), the DCR in IRE-NK (66.7%) was higher than in IRE (42.9%, P < 0.05). Materials and Methods Between July 2016 and May 2017, we enrolled 71 patients who met the enrollment criteria. The patients were divided into stage III (32 patients, 17 patients received only IRE and 15 patients received IRE-NK (Irreversible electroporation- natural killer): 8 patients underwent a course of NK and 7 patients underwent ≥ 3 courses) and stage IV (39 patients, 22 patients received only IRE and 17 patients received IRE-NK: 9 patients underwent a course of NK and 8 patients underwent ≥ 3 courses). The safety and short-term effects were evaluated firstly, then the median PFS, median OS, response rate (RR) and disease control rate (DCR) were assessed. Conclusions Combination of irreversible electroporation and allogeneic natural killer cell immunotherapy significantly increased median PFS and median OS in stage III pancreatic cancer and extended the median OS of stage IV pancreatic cancer. Multiple allogeneic natural killer cells infusion was associated with better prognosis to stage III pancreatic cancer. PMID:29254205

Cell culture imaging using microimpedance tomography.

PubMed

Linderholm, Pontus; Marescot, Laurent; Loke, Meng Heng; Renaud, Philippe

2008-01-01

We present a novel, inexpensive, and fast microimpedance tomography system for two-dimensional imaging of cell and tissue cultures. The system is based on four-electrode measurements using 16 planar microelectrodes (5 microm x 4 mm) integrated into a culture chamber. An Agilent 4294A impedance analyzer combined with a front-end amplifier is used for the impedance measurements. Two-dimensional images are obtained using a reconstruction algorithm. This system is capable of accurately resolving the shape and position of a human hair, yielding vertical cross sections of the object. Human epithelial stem cells (YF 29) are also grown directly on the device surface. Tissue growth can be followed over several days. A rapid resistivity decrease caused by permeabilized cell membranes is also monitored, suggesting that this technique can be used in electroporation studies.

Transient replication of BPV-1 requires two viral polypeptides encoded by the E1 and E2 open reading frames.

PubMed

Ustav, M; Stenlund, A

1991-02-01

Bovine papillomavirus (BPV) DNA is maintained as an episome with a constant copy number in transformed cells and is stably inherited. To study BPV replication we have developed a transient replication assay based on a highly efficient electroporation procedure. Using this assay we have determined that in the context of the viral genome two of the viral open reading frames, E1 and E2, are required for replication. Furthermore we show that when produced from expression vectors in the absence of other viral gene products, the full length E2 transactivator polypeptide and a 72 kd polypeptide encoded by the E1 open reading frame in its entirety, are both necessary and sufficient for replication BPV in C127 cells.

Blood Flow through an Open-Celled Foam

NASA Astrophysics Data System (ADS)

Ortega, Jason; Maitland, Duncan

2011-11-01

The Hazen-Dupuit-Darcy (HDD) equation is commonly used in engineering applications to model the pressure gradient of flow through a porous media. One major advantage of this equation is that it simplifies the complex geometric details of the porous media into two coefficients: the permeability, K, and form factor, C. However through this simplification, the flow details within the porous media are no longer accessible, making it difficult to study the phenomena that contribute to changes in K and C due to clotting of blood flow. To obtain a more detailed understanding of blood flow through a porous media, a direct assessment of the complex interstitial geometry and flow is required. In this study, we solve the Navier-Stokes equations for Newtonian and non-Newtonian blood flow through an open-celled foam geometry obtained from a micro-CT scan. The nominal strut size of the foam sample is of O(10e-5) m and the corresponding Reynolds number based upon this length ranges up to O(10). Fitting the pressure gradient vs. Darcy velocity data with the HDD equation demonstrates that both viscous and inertial forces play an important role in the flow through the foam at these Reynolds numbers. Recirculation zones are observed to form in the wake of the pore struts, producing regions of flow characterized by both low shear rates and long fluid residence times, factors of which have been shown in previous studies to promote blood clotting.

Efficient intracellular delivery and improved biocompatibility of colloidal silver nanoparticles towards intracellular SERS immuno-sensing.

PubMed

Bhardwaj, Vinay; Srinivasan, Supriya; McGoron, Anthony J

2015-06-21

High throughput intracellular delivery strategies, electroporation, passive and TATHA2 facilitated diffusion of colloidal silver nanoparticles (AgNPs) are investigated for cellular toxicity and uptake using state-of-art analytical techniques. The TATHA2 facilitated approach efficiently delivered high payload with no toxicity, pre-requisites for intracellular applications of plasmonic metal nanoparticles (PMNPs) in sensing and therapeutics.

Effects of transgenic sterilization constructs and their repressor compounds on hatch, developmental rate and early survival of electroporated channel catfish embryos and fry.

PubMed

Su, Baofeng; Shang, Mei; Li, Chao; Perera, Dayan A; Pinkert, Carl A; Irwin, Michael H; Peatman, Eric; Grewe, Peter; Patil, Jawahar G; Dunham, Rex A

2015-04-01

Channel catfish (Ictalurus punctatus) embryos were electroporated with sterilization constructs targeting primordial germ cell proteins or with buffer. Some embryos then were treated with repressor compounds, cadmium chloride, copper sulfate, sodium chloride or doxycycline, to prevent expression of the transgene constructs. Promoters included channel catfish nanos and vasa, salmon transferrin (TF), modified yeast Saccharomyces cerevisiae copper transport protein (MCTR) and zebrafish racemase (RM). Knock-down systems were the Tet-off (nanos and vasa constructs), MCTR, RM and TF systems. Knock-down genes included shRNAi targeting 5' nanos (N1), 3' nanos (N2) or dead end (DND), or double-stranded nanos RNA (dsRNA) for overexpression of nanos mRNA. These constructs previously were demonstrated to knock down nanos, vasa and dead end, with the repressors having variable success. Exogenous DNA affected percentage hatch (% hatch), as all 14 constructs, except for the TF dsRNA, TF N1 (T), RM DND (C), vasa DND (C), vasa N1 (C) and vasa N2 (C), had lower % hatch than the control electroporated with buffer. The MCTR and RM DND (T) constructs resulted in delayed hatch, and the vasa and nanos constructs had minimal effects on time of hatch (P < 0.05). Cadmium chloride appeared to counteract the slow development caused by the TF constructs in two TF treatments (P < 0.05). The 4 ppt sodium chloride treatment for the RM system decreased % hatch (P < 0.05) and slowed development. In the case of nanos constructs, doxycycline greatly delayed hatch (P < 0.05). Adverse effects of the transgenes and repressors continued for several treatments for the first 6 days after hatch, but only in a few treatments during the next 10 days. Repressors and gene expression impacted the yield of putative transgenic channel catfish fry, and need to be considered and accounted for in the hatchery phase of producing transgenically sterilized catfish fry and their fertile counterparts. This fry output should be considered to ensure that sufficient numbers of transgenic fish are produced for future applications and for defining repressor systems that are the most successful.

Minimal 2'-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity

PubMed Central

Basila, Megan; Kelley, Melissa L.

2017-01-01

Since its initial application in mammalian cells, CRISPR-Cas9 has rapidly become a preferred method for genome engineering experiments. The Cas9 nuclease is targeted to genomic DNA using guide RNAs (gRNA), either as the native dual RNA system consisting of a DNA-targeting CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), or as a chimeric single guide RNA (sgRNA). Entirely DNA-free CRISPR-Cas9 systems using either Cas9 protein or Cas9 mRNA and chemically synthesized gRNAs allow for transient expression of CRISPR-Cas9 components, thereby reducing the potential for off-targeting, which is a significant advantage in therapeutic applications. In addition, the use of synthetic gRNA allows for the incorporation of chemical modifications for enhanced properties including improved stability. Previous studies have demonstrated the utility of chemically modified gRNAs, but have focused on one pattern with multiple modifications in co-electroporation with Cas9 mRNA or multiple modifications and patterns with Cas9 plasmid lipid co-transfections. Here we present gene editing results using a series of chemically modified synthetic sgRNA molecules and chemically modified crRNA:tracrRNA molecules in both electroporation and lipid transfection assessing indel formation and/or phenotypic gene knockout. We show that while modifications are required for co-electroporation with Cas9 mRNA, some modification patterns of the gRNA are toxic to cells compared to the unmodified gRNA and most modification patterns do not significantly improve gene editing efficiency. We also present modification patterns of the gRNA that can modestly improve Cas9 gene editing efficiency when co-transfected with Cas9 mRNA or Cas9 protein (> 1.5-fold difference). These results indicate that for certain applications, including those relevant to primary cells, the incorporation of some, but not all chemical modification patterns on synthetic crRNA:tracrRNA or sgRNA can be beneficial to CRISPR-Cas9 gene editing. PMID:29176845

Minimal 2'-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity.

PubMed

Basila, Megan; Kelley, Melissa L; Smith, Anja van Brabant

2017-01-01

Since its initial application in mammalian cells, CRISPR-Cas9 has rapidly become a preferred method for genome engineering experiments. The Cas9 nuclease is targeted to genomic DNA using guide RNAs (gRNA), either as the native dual RNA system consisting of a DNA-targeting CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), or as a chimeric single guide RNA (sgRNA). Entirely DNA-free CRISPR-Cas9 systems using either Cas9 protein or Cas9 mRNA and chemically synthesized gRNAs allow for transient expression of CRISPR-Cas9 components, thereby reducing the potential for off-targeting, which is a significant advantage in therapeutic applications. In addition, the use of synthetic gRNA allows for the incorporation of chemical modifications for enhanced properties including improved stability. Previous studies have demonstrated the utility of chemically modified gRNAs, but have focused on one pattern with multiple modifications in co-electroporation with Cas9 mRNA or multiple modifications and patterns with Cas9 plasmid lipid co-transfections. Here we present gene editing results using a series of chemically modified synthetic sgRNA molecules and chemically modified crRNA:tracrRNA molecules in both electroporation and lipid transfection assessing indel formation and/or phenotypic gene knockout. We show that while modifications are required for co-electroporation with Cas9 mRNA, some modification patterns of the gRNA are toxic to cells compared to the unmodified gRNA and most modification patterns do not significantly improve gene editing efficiency. We also present modification patterns of the gRNA that can modestly improve Cas9 gene editing efficiency when co-transfected with Cas9 mRNA or Cas9 protein (> 1.5-fold difference). These results indicate that for certain applications, including those relevant to primary cells, the incorporation of some, but not all chemical modification patterns on synthetic crRNA:tracrRNA or sgRNA can be beneficial to CRISPR-Cas9 gene editing.

Nanosecond pulsed electric fields (nsPEFs) low cost generator design using power MOSFET and Cockcroft-Walton multiplier circuit as high voltage DC source

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sulaeman, M. Y.; Widita, R.

2014-09-30

Purpose: Non-ionizing radiation therapy for cancer using pulsed electric field with high intensity field has become an interesting field new research topic. A new method using nanosecond pulsed electric fields (nsPEFs) offers a novel means to treat cancer. Not like the conventional electroporation, nsPEFs able to create nanopores in all membranes of the cell, including membrane in cell organelles, like mitochondria and nucleus. NsPEFs will promote cell death in several cell types, including cancer cell by apoptosis mechanism. NsPEFs will use pulse with intensity of electric field higher than conventional electroporation, between 20–100 kV/cm and with shorter duration of pulsemore » than conventional electroporation. NsPEFs requires a generator to produce high voltage pulse and to achieve high intensity electric field with proper pulse width. However, manufacturing cost for creating generator that generates a high voltage with short duration for nsPEFs purposes is highly expensive. Hence, the aim of this research is to obtain the low cost generator design that is able to produce a high voltage pulse with nanosecond width and will be used for nsPEFs purposes. Method: Cockcroft-Walton multiplier circuit will boost the input of 220 volt AC into high voltage DC around 1500 volt and it will be combined by a series of power MOSFET as a fast switch to obtain a high voltage with nanosecond pulse width. The motivation using Cockcroft-Walton multiplier is to acquire a low-cost high voltage DC generator; it will use capacitors and diodes arranged like a step. Power MOSFET connected in series is used as voltage divider to share the high voltage in order not to damage them. Results: This design is expected to acquire a low-cost generator that can achieve the high voltage pulse in amount of −1.5 kV with falltime 3 ns and risetime 15 ns into a 50Ω load that will be used for nsPEFs purposes. Further detailed on the circuit design will be explained at presentation.« less

Evaluation of an ARPS-based canopy flow modeling system for use in future operational smoke prediction efforts

Treesearch

M. T. Kiefer; S. Zhong; W. E. Heilman; J. J. Charney; X. Bian

2013-01-01

Efforts to develop a canopy flow modeling system based on the Advanced Regional Prediction System (ARPS) model are discussed. The standard version of ARPS is modified to account for the effect of drag forces on mean and turbulent flow through a vegetation canopy, via production and sink terms in the momentum and subgrid-scale turbulent kinetic energy (TKE) equations....

An inverse method to estimate the flow through a levee breach

NASA Astrophysics Data System (ADS)

D'Oria, Marco; Mignosa, Paolo; Tanda, Maria Giovanna

2015-08-01

We propose a procedure to estimate the flow through a levee breach based on water levels recorded in river stations downstream and/or upstream of the failure site. The inverse problem is solved using a Bayesian approach and requires the execution of several forward unsteady flow simulations. For this purpose, we have used the well-known 1-D HEC-RAS model, but any unsteady flow model could be adopted in the same way. The procedure has been tested using four synthetic examples. Levee breaches with different characteristics (free flow, flow with tailwater effects, etc.) have been simulated to collect the synthetic level data used at a later stage in the inverse procedure. The method was able to accurately reproduce the flow through the breach in all cases. The practicability of the procedure was then confirmed applying it to the inundation of the Polesine Region (Northern Italy) which occurred in 1951 and was caused by three contiguous and almost simultaneous breaches on the left embankment of the Po River.

Transformation-Induced, Geometrically Necessary, Dislocation-Based Flow Curve Modeling of Dual-Phase Steels: Effect of Grain Size

NASA Astrophysics Data System (ADS)

Ramazani, Ali; Mukherjee, Krishnendu; Prahl, Ulrich; Bleck, Wolfgang

2012-10-01

The flow behavior of dual-phase (DP) steels is modeled on the finite-element method (FEM) framework on the microscale, considering the effect of the microstructure through the representative volume element (RVE) approach. Two-dimensional RVEs were created from microstructures of experimentally obtained DP steels with various ferrite grain sizes. The flow behavior of single phases was modeled through the dislocation-based work-hardening approach. The volume change during austenite-to-martensite transformation was modeled, and the resultant prestrained areas in the ferrite were considered to be the storage place of transformation-induced, geometrically necessary dislocations (GNDs). The flow curves of DP steels with varying ferrite grain sizes, but constant martensite fractions, were obtained from the literature. The flow curves of simulations that take into account the GND are in better agreement with those of experimental flow curves compared with those of predictions without consideration of the GND. The experimental results obeyed the Hall-Petch relationship between yield stress and flow stress and the simulations predicted this as well.

Ferromagnetic effects for nanofluid venture through composite permeable stenosed arteries with different nanosize particles

NASA Astrophysics Data System (ADS)

Akbar, Noreen Sher; Mustafa, M. T.

2015-07-01

In the present article ferromagnetic field effects for copper nanoparticles for blood flow through composite permeable stenosed arteries is discussed. The copper nanoparticles for the blood flow with water as base fluid with different nanosize particles is not explored upto yet. The equations for the Cu-water nanofluid are developed first time in literature and simplified using long wavelength and low Reynolds number assumptions. Exact solutions have been evaluated for velocity, pressure gradient, the solid volume fraction of the nanoparticles and temperature profile. Effect of various flow parameters on the flow and heat transfer characteristics are utilized.

Deformation of an elastic capsule in a uniform electric field

NASA Astrophysics Data System (ADS)

Karyappa, Rahul B.; Deshmukh, Shivraj. D.; Thaokar, Rochish. M.

2014-12-01

The deformation of a thin elastic capsule subjected to a uniform electric field is investigated in the Stokes flow regime. The electrohydrodynamic flow is analyzed using a perfect conductor and a perfect dielectric model for the capsule and the fluid phase, respectively. A theoretical analysis is carried out using an asymptotic expansion in the electric capillary number (Ca) (a ratio of the electric stress to the elastic tension) in the small deformation limit using the finite deformation Hooke's law. The analysis is used to determine the elasticity of polysiloxane capsules suspended in oil, the deformation of which is obtained using videography. The boundary element method is implemented to seek numerical solutions to the hydrodynamic, elastic, and electrostatics equations. The finite deformation Hooke's law, the Mooney-Rivlin, and Skalak's model for elasticity are employed. The effect of electric capillary number, unstressed geometry, and the type of membrane material on the deformation of a capsule is presented in the high Ca number limit using numerical simulation. Capsules synthesized with higher monomer concentration displayed electric stress induced wrinkling process at high electric field strengths. Burst of a capsule is characterized by poration of the polymer membrane, which could be symmetric or asymmetric at the two poles, depending upon the value of the capillary number. The results should be useful in understanding the response of elastic capsules such as red blood cells and polymerized membranes, to an electric field, in applications such as electrodeformation and electroporation. It also provides a theoretical framework for a possible way of determining the elastic parameters of a capsule.

Climate change could drive marine food web collapse through altered trophic flows and cyanobacterial proliferation

PubMed Central

Ullah, Hadayet; Goldenberg, Silvan U.; Fordham, Damien A.

2018-01-01

Global warming and ocean acidification are forecast to exert significant impacts on marine ecosystems worldwide. However, most of these projections are based on ecological proxies or experiments on single species or simplified food webs. How energy fluxes are likely to change in marine food webs in response to future climates remains unclear, hampering forecasts of ecosystem functioning. Using a sophisticated mesocosm experiment, we model energy flows through a species-rich multilevel food web, with live habitats, natural abiotic variability, and the potential for intra- and intergenerational adaptation. We show experimentally that the combined stress of acidification and warming reduced energy flows from the first trophic level (primary producers and detritus) to the second (herbivores), and from the second to the third trophic level (carnivores). Warming in isolation also reduced the energy flow from herbivores to carnivores, the efficiency of energy transfer from primary producers and detritus to herbivores and detritivores, and the living biomass of detritivores, herbivores, and carnivores. Whilst warming and acidification jointly boosted primary producer biomass through an expansion of cyanobacteria, this biomass was converted to detritus rather than to biomass at higher trophic levels—i.e., production was constrained to the base of the food web. In contrast, ocean acidification affected the food web positively by enhancing trophic flow from detritus and primary producers to herbivores, and by increasing the biomass of carnivores. Our results show how future climate change can potentially weaken marine food webs through reduced energy flow to higher trophic levels and a shift towards a more detritus-based system, leading to food web simplification and altered producer–consumer dynamics, both of which have important implications for the structuring of benthic communities. PMID:29315309

MODELING AIR FLOW DYNAMICS IN RADON MITIGATION SYSTEMS: A SIMPLIFIED APPROACH

EPA Science Inventory

The paper refines and extends an earlier study--relating to the design of optimal radon mitigation systems based on subslab depressurization-- that suggested that subslab air flow induced by a central suction point be treated as radial air flow through a porous bed contained betw...

Mapping debris-flow hazard in Honolulu using a DEM

USGS Publications Warehouse

Ellen, Stephen D.; Mark, Robert K.; ,

1993-01-01

A method for mapping hazard posed by debris flows has been developed and applied to an area near Honolulu, Hawaii. The method uses studies of past debris flows to characterize sites of initiation, volume at initiation, and volume-change behavior during flow. Digital simulations of debris flows based on these characteristics are then routed through a digital elevation model (DEM) to estimate degree of hazard over the area.

How Important Is Connectivity for Surface Water Fluxes? A Generalized Expression for Flow Through Heterogeneous Landscapes

NASA Astrophysics Data System (ADS)

Larsen, Laurel G.; Ma, Jie; Kaplan, David

2017-10-01

How important is hydrologic connectivity for surface water fluxes through heterogeneous floodplains, deltas, and wetlands? While significant for management, this question remains poorly addressed. Here we adopt spatial resistance averaging, based on channel and patch configuration metrics quantifiable from aerial imagery, to produce an upscaled rate law for discharge. Our model suggests that patch coverage largely controls discharge sensitivity, with smaller effects from channel connectivity and vegetation patch fractal dimension. However, connectivity and patch configuration become increasingly important near the percolation threshold and at low water levels. These effects can establish positive feedbacks responsible for substantial flow change in evolving landscapes (14-36%, in our Everglades case study). Connectivity also interacts with other drivers; flow through poorly connected hydroscapes is less resilient to perturbations in other drivers. Finally, we found that flow through heterogeneous patches is alone sufficient to produce non-Manning flow-depth relationships commonly observed in wetlands but previously attributed to depth-varying roughness.

A Supersonic Tunnel for Laser and Flow-Seeding Techniques

NASA Technical Reports Server (NTRS)

Bruckner, Robert J.; Lepicovsky, Jan

1994-01-01

A supersonic wind tunnel with flow conditions of 3 lbm/s (1.5 kg/s) at a free-stream Mach number of 2.5 was designed and tested to provide an arena for future development work on laser measurement and flow-seeding techniques. The hybrid supersonic nozzle design that was used incorporated the rapid expansion method of propulsive nozzles while it maintained the uniform, disturbance-free flow required in supersonic wind tunnels. A viscous analysis was performed on the tunnel to determine the boundary layer growth characteristics along the flowpath. Appropriate corrections were then made to the contour of the nozzle. Axial pressure distributions were measured and Mach number distributions were calculated based on three independent data reduction methods. A complete uncertainty analysis was performed on the precision error of each method. Complex shock-wave patterns were generated in the flow field by wedges mounted near the roof and floor of the tunnel. The most stable shock structure was determined experimentally by the use of a focusing schlieren system and a novel, laser based dynamic shock position sensor. Three potential measurement regions for future laser and flow-seeding studies were created in the shock structure: deceleration through an oblique shock wave of 50 degrees, strong deceleration through a normal shock wave, and acceleration through a supersonic expansion fan containing 25 degrees of flow turning.

Development of flow-through and dip-stick immunoassays for screening of sulfonamide residues.

PubMed

Zhang, Hongyan; Zhang, Yan; Wang, Shuo

2008-08-20

Two formats of membrane-based competitive enzyme immunoassays (flow-through and dip-stick) have been developed for the screening of sulfonamide residues in pig muscle and milk. Membrane was coated with anti-sulfonamide antibody and a sulfonamide hapten D2-horseradish peroxidase (HRP) conjugant was used as the labeled antigen for competitive assay of sulfonamides. Visual detection limits of the flow-through or dip-stick assay were 1-5 microg L(-1) or 1-10 microg L(-1) in buffer for seven sulfonamides, respectively. Assay validation was performed using samples spiked with single sulfonamide, spiked samples were tested using the developed strip assays and results were compared with those obtained by a validated high-performance liquid chromatograph (HPLC) method. Results showed that the two strip assays were correlated well with HPLC, respectively. With assay times of 5 min (flow-through) and 15 min (dip-stick), these rapid tests could offer simple, rapid and cost-effective on-site screening tools to detect sulfonamides in pig muscle (flow-through or dip-stick) or milk (only dip-stick).

A Capillary Flow Dynamics-Based Sensing Modality for Direct Environmental Pathogen Monitoring.

PubMed

Klug, Katherine E; Reynolds, Kelly A; Yoon, Jeong-Yeol

2018-04-20

Toward ultra-simple and field-ready biosensors, we demonstrate a novel assay transducer mechanism based on interfacial property changes and capillary flow dynamics in antibody-conjugated submicron particle suspensions. Differential capillary flow is tunable, allowing pathogen quantification as a function of flow rate through a paper-based microfluidic device. Flow models based on interfacial and rheological properties indicate a significant relationship between the flow rate and the interfacial effects caused by target-particle aggregation. This mechanism is demonstrated for assays of Escherichia coli K12 in water samples and Zika virus (ZIKV) in blood serum. These assays achieved very low limits of detection compared with other demonstrated methods (1 log CFU/mL E. coli and 20 pg/mL ZIKV whole virus) with an operating time of 30 s, showing promise for environmental and health monitoring. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Road crossings as barriers to small-stream fish movement

Treesearch

Melvin L. Warren; Mitzi G. Pardew

1998-01-01

The authors used mark-recapture techniques to examine the effects of four types of road crossings on fish movement during spring base flows and summer low flows in small streams of the Ouachita Mountains, west-central Arkansas. The authors assessed movement for 21 fish species in seven families through culvert, slab, open-box, and ford crossings and through natural...

Three-dimensional turbopump flowfield analysis

NASA Technical Reports Server (NTRS)

Sharma, O. P.; Belford, K. A.; Ni, R. H.

1992-01-01

A program was conducted to develop a flow prediction method applicable to rocket turbopumps. The complex nature of a flowfield in turbopumps is described and examples of flowfields are discussed to illustrate that physics based models and analytical calculation procedures based on computational fluid dynamics (CFD) are needed to develop reliable design procedures for turbopumps. A CFD code developed at NASA ARC was used as the base code. The turbulence model and boundary conditions in the base code were modified, respectively, to: (1) compute transitional flows and account for extra rates of strain, e.g., rotation; and (2) compute surface heat transfer coefficients and allow computation through multistage turbomachines. Benchmark quality data from two and three-dimensional cascades were used to verify the code. The predictive capabilities of the present CFD code were demonstrated by computing the flow through a radial impeller and a multistage axial flow turbine. Results of the program indicate that the present code operated in a two-dimensional mode is a cost effective alternative to full three-dimensional calculations, and that it permits realistic predictions of unsteady loadings and losses for multistage machines.

The shape and motion of gas bubbles in a liquid flowing through a thin annulus

NASA Astrophysics Data System (ADS)

Lei, Qinghua; Xie, Zhihua; Pavlidis, Dimitrios; Salinas, Pablo; Veltin, Jeremy; Muggeridge, Ann; Pain, Christopher C.; Matar, Omar K.; Jackson, Matthew; Arland, Kristine; Gyllensten, Atle

2017-11-01

We study the shape and motion of gas bubbles in a liquid flowing through a horizontal or slightly-inclined thin annulus. Experimental data show that in the horizontal annulus, bubbles develop a unique ``tadpole'' shape with an elliptical cap and a highly-stretched tail, due to the confinement between the closely-spaced channel walls. As the annulus is inclined, the bubble tail tends to decrease in length, while the geometry of the cap remains almost invariant. To model the bubble evolution, the thin annulus is conceptualised as a ``Hele-Shaw'' cell in a curvilinear space. The three-dimensional flow within the cell is represented by a gap-averaged, two-dimensional model constrained by the same dimensionless quantities. The complex bubble dynamics are solved using a mixed control-volume finite-element method combined with interface-capturing and mesh adaptation techniques. A close match to the experimental data is achieved, both qualitatively and quantitatively, by the numerical simulations. The mechanism for the elliptical cap formation is interpreted based on an analogous irrotational flow field around a circular cylinder. The shape regimes of bubbles flowing through the thin annulus are further explored based on the simulation results. Funding from STATOIL gratefully acknowledged.

DNA Vaccine Electroporation and Molecular Adjuvants

DTIC Science & Technology

2016-03-16

lethal species with a mortality range of 60-90% in human outbreaks [2]. The Marburgvirus genus currently includes a single viral species, Marburg...single viral species, Lloviu cuevavirus, and one defined virus, Lloviu virus (LLOV). Due to the high mortality rate, person to person TR-16-113...correlates of protective immunity, but progress has recently been made. The viral envelope glycoprotein, GP, is the main target of antibody responses [1

Investigation of Mixing a Supersonic Stream with the Flow Downstream of a Wedge

NASA Technical Reports Server (NTRS)

Sheeley, Joseph

1997-01-01

The flow characteristics in the base region of a two-dimensional supersonic compression ramp are investigated. A stream-wise oriented air jet, M = 1.75, is injected through a thin horizontal slot into a supersonic air main flow, M = 2.3, at the end of a two-dimensional compression ramp. The velocity profile and basic characteristics of the flow in the base region immediately following the ramp are determined. Visualization of the flowfield for qualitative observations is accomplished via Dark Central Ground Interferometry (DCGI). Two-dimensional velocity profiles are obtained using Laser Doppler Velocimetry (LDV). The study is the initial phase of a four-year investigation of base flow mixing. The current study is to provide more details of the flowfield.

Irreversible Electroporation of Human Primary Uveal Melanoma in Enucleated Eyes

PubMed Central

Mandel, Yossi; Laufer, Shlomi; Belkin, Michael; Rubinsky, Boris; Pe'er, Jacob; Frenkel, Shahar

2013-01-01

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. PMID:24039721

A novel method for the production of transgenic cloned pigs: electroporation-mediated gene transfer to non-cultured cells and subsequent selection with puromycin.

PubMed

Watanabe, Satoshi; Iwamoto, Masaki; Suzuki, Shun-ichi; Fuchimoto, Daiichiro; Honma, Daisuke; Nagai, Takashi; Hashimoto, Michiko; Yazaki, Satoko; Sato, Masahiro; Onishi, Akira

2005-02-01

Puromycin N-acetyl transferase gene (pac), of which the gene product catalyzes antibiotic puromycin (an effective inhibitor of protein synthesis), has been widely used as a dominant selection marker in embryonic stem (ES) cell-mediated transgenesis. The present study is the first to report on the usefulness of puromycin for production of enhanced green fluorescent protein (EGFP) transgenic piglets after somatic cell cloning and embryo transfer. Somatic cells isolated from porcine fetuses at 73 days of gestation were immediately electroporated with a transgene (pCAG-EGFPac) carrying both EGFP cDNA and pac. This procedure aims to avoid aging effects thought to be generated during cell culture. The recombinant cells were selected with puromycin at a low concentration (2 microg/ml), cultured for 7 days, and then screened for EGFP expression before somatic cell cloning. The manipulated embryos were transplanted into the oviducts of 14 foster mother sows. Four of the foster sows became pregnant and nine piglets were delivered. Of the nine piglets, eight died shortly after birth and one grew healthy after weaning. Results indicate that puromycin can be used for the selection of recombinant cells from noncultured cells, and moreover, may confer the production of genetically engineered newborns via nuclear transfer techniques in pigs.

Electrical impedance characterization of normal and cancerous human hepatic tissue.

PubMed

Laufer, Shlomi; Ivorra, Antoni; Reuter, Victor E; Rubinsky, Boris; Solomon, Stephen B

2010-07-01

The four-electrode method was used to measure the ex vivo complex electrical impedance of tissues from 14 hepatic tumors and the surrounding normal liver from six patients. Measurements were done in the frequency range 1-400 kHz. It was found that the conductivity of the tumor tissue was much higher than that of the normal liver tissue in this frequency range (from 0.14 +/- 0.06 S m(-1) versus 0.03 +/- 0.01 S m(-1) at 1 kHz to 0.25 +/- 0.06 S m(-1) versus 0.15 +/- 0.03 S m(-1) at 400 kHz). The Cole-Cole models were estimated from the experimental data and the four parameters (rho(0), rho(infinity), alpha, f(c)) were obtained using a least-squares fit algorithm. The Cole-Cole parameters for the cancerous and normal liver are 9 +/- 4 Omega m(-1), 2.2 +/- 0.7 Omega m(-1), 0.5 +/- 0.2, 140 +/- 103 kHz and 50 +/- 28 Omega m(-1), 3.2 +/- 0.6 Omega m(-1), 0.64 +/- 0.04, 10 +/- 7 kHz, respectively. These data can contribute to developing bioelectric applications for tissue diagnostics and in tissue treatment planning with electrical fields such as radiofrequency tissue ablation, electrochemotherapy and gene therapy with reversible electroporation, nanoscale pulsing and irreversible electroporation.

Clinical and pathological outcomes after irreversible electroporation of the pancreas using two parallel plate electrodes: a porcine model.

PubMed

Rombouts, Steffi J E; van Dijck, Willemijn P M; Nijkamp, Maarten W; Derksen, Tyche C; Brosens, Lodewijk A A; Hoogwater, Frederik J H; van Leeuwen, Maarten S; Borel Rinkes, Inne H M; van Hillegersberg, Richard; Wittkampf, Fred H; Molenaar, Izaak Q

2017-12-01

Irreversible electroporation (IRE) by inserting needles around the tumor as treatment for locally advanced pancreatic cancer entails several disadvantages, such as incomplete ablation due to field inhomogeneity, technical difficulties in needle placement and a risk of pancreatic fistula development. This experimental study evaluates outcomes of IRE using paddles in a porcine model. Six healthy pigs underwent laparotomy and were treated with 2 separate ablations (in head and tail of the pancreas). Follow-up consisted of clinical and laboratory parameters and contrast-enhanced computed tomography (ceCT) imaging. After 2 weeks, pancreatoduodenectomy was performed for histology and the pigs were terminated. All animals survived 14 days. None of the animals developed signs of infection or significant abdominal distention. Serum amylase and lipase peaked at day 1 postoperatively in all pigs, but normalized without signs of pancreatitis. On ceCT-imaging the ablation zone was visible as an ill-defined, hypodense lesion. No abscesses, cysts or ascites were seen. Histology showed a homogenous fibrotic lesion in all pigs. IRE ablation of healthy porcine pancreatic tissue using two plate electrodes is feasible and safe and creates a homogeneous fibrotic lesion. IRE-paddles should be tested on pancreatic adenocarcinoma to determine the effect in cancer tissue. Copyright © 2017. Published by Elsevier Ltd.

In situ formation of magnetopolymersomes via electroporation for MRI

NASA Astrophysics Data System (ADS)

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.

Electric Ablation with Irreversible Electroporation (IRE) in Vital Hepatic Structures and Follow-up Investigation

PubMed Central

Chen, Xinhua; Ren, Zhigang; Zhu, Tongyin; Zhang, Xiongxin; Peng, Zhiyi; Xie, Haiyang; Zhou, Lin; Yin, Shengyong; Sun, Junhui; Zheng, Shusen

2015-01-01

Irreversible electroporation (IRE) with microsecond-pulsed electric fields (μsPEFs) can effectively ablate hepatocellular carcinomas in animal models. This preclinical study evaluates the feasibility and safety of IRE on porcine livers. Altogether, 10 pigs were included. Computed tomography (CT) was used to guide two-needle electrodes that were inserted near the hilus hepatis and gall bladder. Animals were followed-up at 2 hours and at 2, 7 and 14 days post-treatment. During and after μsPEF ablation, electrocardiographs found no cardiovascular events, and contrast CT found no portal vein thrombosis. There was necrosis in the ablation zone. Mild cystic oedema around the gall bladder was found 2 hours post-treatment. Pathological studies showed extensive cell death. There was no large vessel damage, but there was mild endothelial damage in some small vessels. Follow-up liver function tests and routine blood tests showed immediate liver function damage and recovery from the damage, which correlated to the pathological changes. These results indicate that μsPEF ablation affects liver tissue and is less effective in vessels, which enable μsPEFs to ablate central tumour lesions close to the hilus hepatis and near large vessels and bile ducts, removing some of the limitations and contraindications of conventional thermal ablation. PMID:26549662

Pathology of non-thermal irreversible electroporation (N-TIRE)-induced ablation of the canine brain.

PubMed

Rossmeisl, John H; Garcia, Paulo A; Roberston, John L; Ellis, Thomas L; Davalos, Rafael V

2013-01-01

This study describes the neuropathologic features of normal canine brain ablated with non-thermal irreversible electroporation (N-TIRE). The parietal cerebral cortices of four dogs were treated with N-TIRE using a dose-escalation protocol with an additional dog receiving sham treatment. Animals were allowed to recover following N-TIRE ablation and the effects of treatment were monitored with clinical and magnetic resonance imaging examinations. Brains were subjected to histopathologic and ultrastructural assessment along with Bcl-2, caspase-3, and caspase-9 immunohistochemical staining following sacrifice 72 h post-treatment. Adverse clinical effects of N-TIRE were only observed in the dog treated at the upper energy tier. MRI and neuropathologic examinations indicated that N-TIRE ablation resulted in focal regions of severe cytoarchitectural and blood-brain-barrier disruption. Lesion size correlated to the intensity of the applied electrical field. N-TIRE-induced lesions were characterized by parenchymal necrosis and hemorrhage; however, large blood vessels were preserved. A transition zone containing parenchymal edema, perivascular inflammatory cuffs, and reactive gliosis was interspersed between the necrotic focus and normal neuropil. Apoptotic labeling indices were not different between the N-TIRE-treated and control brains. This study identified N-TIRE pulse parameters that can be used to safely create circumscribed foci of brain necrosis while selectively preserving major vascular structures.

Irreversible electroporation of the pancreas is feasible and safe in a porcine survival model.

PubMed

Fritz, Stefan; Sommer, Christof M; Vollherbst, Dominik; Wachter, Miguel F; Longerich, Thomas; Sachsenmeier, Milena; Knapp, Jürgen; Radeleff, Boris A; Werner, Jens

2015-07-01

Use of thermal tumor ablation in the pancreatic parenchyma is limited because of the risk of pancreatitis, pancreatic fistula, or hemorrhage. This study aimed to evaluate the feasibility and safety of irreversible electroporation (IRE) in a porcine model. Ten pigs were divided into 2 study groups. In the first group, animals received IRE of the pancreatic tail and were killed after 60 minutes. In the second group, animals received IRE at the head of the pancreas and were followed up for 7 days. Clinical parameters, computed tomography imaging, laboratory results, and histology were obtained. All animals survived IRE ablation, and no cardiac adverse effects were noted. Sixty minutes after IRE, a hypodense lesion on computed tomography imaging indicated the ablation zone. None of the animals developed clinical signs of acute pancreatitis. Only small amounts of ascites fluid, with a transient increase in amylase and lipase levels, were observed, indicating that no pancreatic fistula occurred. This porcine model shows that IRE is feasible and safe in the pancreatic parenchyma. Computed tomography imaging reveals significant changes at 60 minutes after IRE and therefore might serve as an early indicator of therapeutic success. Clinical studies are needed to evaluate the efficacy of IRE in pancreatic cancer.

Experimental characterization of intrapulse tissue conductivity changes for electroporation.

PubMed

Neal, Robert E; Garcia, Paulo A; Robertson, John L; Davalos, Rafael V

2011-01-01

Cells exposed to short electric pulses experience a change in their transmembrane potential, which can lead to increased membrane permeability of the cell. When the energy of the pulses surpasses a threshold, the cell dies in a non-thermal manner known as irreversible electroporation (IRE). IRE has shown promise in the focal ablation of pathologic tissues. Its non-thermal mechanism spares sensitive structures and facilitates rapid lesion resolution. IRE effects depend on the electric field distribution, which can be predicted with numerical modeling. When the cells become permeabilized, the bulk tissue properties change, affecting this distribution. For IRE to become a reliable and successful treatment of diseased tissues, robust predictive treatment planning methods must be developed. It is vital to understand the changes in tissue properties undergoing the electric pulses to improve numerical models and predict treatment volumes. We report on the experimental characterization of these changes for kidney tissue. Tissue samples were pulsed between plate electrodes while intrapulse voltage and current data were measured to determine the conductivity of the tissue during the pulse. Conductivity was then established as a function of the electric field to which the tissue is exposed. This conductivity curve was used in a numerical model to demonstrate the impact of accounting for these changes when modeling electric field distributions to develop treatment plans.

An improved yeast transformation method for the generation of very large human antibody libraries.

PubMed

Benatuil, Lorenzo; Perez, Jennifer M; Belk, Jonathan; Hsieh, Chung-Ming

2010-04-01

Antibody library selection by yeast display technology is an efficient and highly sensitive method to identify binders to target antigens. This powerful selection tool, however, is often hampered by the typically modest size of yeast libraries (approximately 10(7)) due to the limited yeast transformation efficiency, and the full potential of the yeast display technology for antibody discovery and engineering can only be realized if it can be coupled with a mean to generate very large yeast libraries. We describe here a yeast transformation method by electroporation that allows for the efficient generation of large antibody libraries up to 10(10) in size. Multiple components and conditions including CaCl(2), MgCl(2), sucrose, sorbitol, lithium acetate, dithiothreitol, electroporation voltage, DNA input and cell volume have been tested to identify the best combination. By applying this developed protocol, we have constructed a 1.4 x 10(10) human spleen antibody library essentially in 1 day with a transformation efficiency of 1-1.5 x 10(8) transformants/microg vector DNA. Taken together, we have developed a highly efficient yeast transformation method that enables the generation of very large and productive human antibody libraries for antibody discovery, and we are now routinely making 10(9) libraries in a day for antibody engineering purposes.

Transposon mutagenesis of Xylella fastidiosa by electroporation of Tn5 synaptic complexes.

PubMed

Guilhabert, M R; Hoffman, L M; Mills, D A; Kirkpatrick, B C

2001-06-01

Pierce's disease, a lethal disease of grapevine, is caused by Xylella fastidiosa, a gram-negative, xylem-limited bacterium that is transmitted from plant to plant by xylem-feeding insects. Strains of X. fastidiosa also have been associated with diseases that cause tremendous losses in many other economically important plants, including citrus. Although the complete genome sequence of X. fastidiosa has recently been determined, the inability to transform or produce transposon mutants of X. fastidiosa has been a major impediment to understanding pathogen-, plant-, and insect-vector interactions. We evaluated the ability of four different suicide vectors carrying either Tn5 or Tn10 transposons as well as a preformed Tn5 transposase-transposon synaptic complex (transposome) to transpose X. fastidiosa. The four suicide vectors failed to produce any detectable transposition events. Electroporation of transposomes, however, yielded 6 x 10(3) and 4 x 10(3) Tn5 mutants per microg of DNA in two different grapevine strains of X. fastidiosa. Molecular analysis showed that the transposition insertions were single, independent, stable events. Sequence analysis of the Tn5 insertion sites indicated that the transpositions occur randomly in the X. fastidiosa genome. Transposome-mediated mutagenesis should facilitate the identification of X. fastidiosa genes that mediate plant pathogenicity and insect transmission.

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

PubMed Central

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

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

PubMed

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

2016-10-26

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.

A simple method for multiday imaging of slice cultures.

PubMed

Seidl, Armin H; Rubel, Edwin W

2010-01-01

The organotypic slice culture (Stoppini et al. A simple method for organotypic cultures of nervous tissue. 1991;37:173-182) has become the method of choice to answer a variety of questions in neuroscience. For many experiments, however, it would be beneficial to image or manipulate a slice culture repeatedly, for example, over the course of many days. We prepared organotypic slice cultures of the auditory brainstem of P3 and P4 mice and kept them in vitro for up to 4 weeks. Single cells in the auditory brainstem were transfected with plasmids expressing fluorescent proteins by way of electroporation (Haas et al. Single-cell electroporation for gene transfer in vivo. 2001;29:583-591). The culture was then placed in a chamber perfused with oxygenated ACSF and the labeled cell imaged with an inverted wide-field microscope repeatedly for multiple days, recording several time-points per day, before returning the slice to the incubator. We describe a simple method to image a slice culture preparation during the course of multiple days and over many continuous hours, without noticeable damage to the tissue or photobleaching. Our method uses a simple, inexpensive custom-built insulator constructed around the microscope to maintain controlled temperature and uses a perfusion chamber as used for in vitro slice recordings. (c) 2009 Wiley-Liss, Inc.

Electric Ablation with Irreversible Electroporation (IRE) in Vital Hepatic Structures and Follow-up Investigation.

PubMed

Chen, Xinhua; Ren, Zhigang; Zhu, Tongyin; Zhang, Xiongxin; Peng, Zhiyi; Xie, Haiyang; Zhou, Lin; Yin, Shengyong; Sun, Junhui; Zheng, Shusen

2015-11-09

Irreversible electroporation (IRE) with microsecond-pulsed electric fields (μsPEFs) can effectively ablate hepatocellular carcinomas in animal models. This preclinical study evaluates the feasibility and safety of IRE on porcine livers. Altogether, 10 pigs were included. Computed tomography (CT) was used to guide two-needle electrodes that were inserted near the hilus hepatis and gall bladder. Animals were followed-up at 2 hours and at 2, 7 and 14 days post-treatment. During and after μsPEF ablation, electrocardiographs found no cardiovascular events, and contrast CT found no portal vein thrombosis. There was necrosis in the ablation zone. Mild cystic oedema around the gall bladder was found 2 hours post-treatment. Pathological studies showed extensive cell death. There was no large vessel damage, but there was mild endothelial damage in some small vessels. Follow-up liver function tests and routine blood tests showed immediate liver function damage and recovery from the damage, which correlated to the pathological changes. These results indicate that μsPEF ablation affects liver tissue and is less effective in vessels, which enable μsPEFs to ablate central tumour lesions close to the hilus hepatis and near large vessels and bile ducts, removing some of the limitations and contraindications of conventional thermal ablation.

Incompressible viscous flow computations for the pump components and the artificial heart

NASA Technical Reports Server (NTRS)

Kiris, Cetin

1992-01-01

A finite-difference, three-dimensional incompressible Navier-Stokes formulation to calculate the flow through turbopump components is utilized. The solution method is based on the pseudocompressibility approach and uses an implicit-upwind differencing scheme together with the Gauss-Seidel line relaxation method. Both steady and unsteady flow calculations can be performed using the current algorithm. In this work, the equations are solved in steadily rotating reference frames by using the steady-state formulation in order to simulate the flow through a turbopump inducer. Eddy viscosity is computed by using an algebraic mixing-length turbulence model. Numerical results are compared with experimental measurements and a good agreement is found between the two. Included in the appendix is a paper on incompressible viscous flow through artificial heart devices with moving boundaries. Time-accurate calculations, such as impeller and diffusor interaction, will be reported in future work.

Stochastic cycle selection in active flow networks

NASA Astrophysics Data System (ADS)

Woodhouse, Francis; Forrow, Aden; Fawcett, Joanna; Dunkel, Jorn

2016-11-01

Active biological flow networks pervade nature and span a wide range of scales, from arterial blood vessels and bronchial mucus transport in humans to bacterial flow through porous media or plasmodial shuttle streaming in slime molds. Despite their ubiquity, little is known about the self-organization principles that govern flow statistics in such non-equilibrium networks. By connecting concepts from lattice field theory, graph theory and transition rate theory, we show how topology controls dynamics in a generic model for actively driven flow on a network. Through theoretical and numerical analysis we identify symmetry-based rules to classify and predict the selection statistics of complex flow cycles from the network topology. Our conceptual framework is applicable to a broad class of biological and non-biological far-from-equilibrium networks, including actively controlled information flows, and establishes a new correspondence between active flow networks and generalized ice-type models.

Non-Darcian flow of shear-thinning fluids through packed beads: Experiments and predictions using Forchheimer's law and Ergun's equation

NASA Astrophysics Data System (ADS)

Rodríguez de Castro, Antonio; Radilla, Giovanni

2017-02-01

The flow of shear-thinning fluids through unconsolidated porous media is present in a number of important industrial applications such as soil depollution, Enhanced Oil Recovery or filtration of polymeric liquids. Therefore, predicting the pressure drop-flow rate relationship in model porous media has been the scope of major research efforts during the last decades. Although the flow of Newtonian fluids through packs of spherical particles is well understood in most cases, much less is known regarding the flow of shear-thinning fluids as high molecular weight polymer aqueous solutions. In particular, the experimental data for the non-Darcian flow of shear-thinning fluids are scarce and so are the current approaches for their prediction. Given the relevance of non-Darcian shear-thinning flow, the scope of this work is to perform an experimental study to systematically evaluate the effects of fluid shear rheology on the flow rate-pressure drop relationships for the non-Darcian flow through different packs of glass spheres. To do so, xanthan gum aqueous solutions with different polymer concentrations are injected through four packs of glass spheres with uniform size under Darcian and inertial flow regimes. A total of 1560 experimental data are then compared with predictions coming from different methods based on the extension of widely used Ergun's equation and Forchheimer's law to the case of shear thinning fluids, determining the accuracy of these predictions. The use of a proper definition for Reynolds number and a realistic model to represent the rheology of the injected fluids results in the porous media are shown to be key aspects to successfully predict pressure drop-flow rate relationships for the inertial shear-thinning flow in packed beads.

Development of cadmium-free quantum dot for intracellular labelling through electroporation or lipid-calcium-phosphate

NASA Astrophysics Data System (ADS)

Liu, Ying-Feng; Hung, Wei-Ling; Hou, Tzh-Yin; Huang, Hsiu-Ying; Lin, Cheng-An J.

2016-04-01

Traditional fluorescent labelling techniques has severe photo-bleaching problem such as organic dyes and fluorescent protein. Quantum dots made up of traditional semiconductor (CdSe/ZnS) material has sort of biological toxicity. This research has developed novel Cd-free quantum dots divided into semiconductor (Indium phosphide, InP) and noble metal (Gold). Former has lower toxicity compared to traditional quantum dots. Latter consisting of gold (III) chloride (AuCl3) and toluene utilizes sonochemical preparation and different stimulus to regulate fluorescent wavelength. Amphoteric macromolecule surface technology and ligand Exchange in self-Assembled are involved to develop hydrophilic nanomaterials which can regulate the number of grafts per molecule of surface functional groups. Calcium phosphate (CaP) nanoparticle (NP) with an asymmetric lipid bilayer coating technology developed for intracellular delivery and labelling has synthesized Cd-free quantum dots possessing high brightness and multi-fluorescence successfully. Then, polymer coating and ligand exchange transfer to water-soluble materials to produce liposome nanomaterials as fluorescent probes and enhancing medical applications of nanotechnology.

Gene Electrotransfer: A Mechanistic Perspective

PubMed Central

Rosazza, Christelle; Meglic, Sasa Haberl; Zumbusch, Andreas; Rols, Marie-Pierre; Miklavcic, Damijan

2016-01-01

Gene electrotransfer is a powerful method of DNA delivery offering several medical applications, among the most promising of which are DNA vaccination and gene therapy for cancer treatment. Electroporation entails the application of electric fields to cells which then experience a local and transient change of membrane permeability. Although gene electrotransfer has been extensively studied in in vitro and in vivo environments, the mechanisms by which DNA enters and navigates through cells are not fully understood. Here we present a comprehensive review of the body of knowledge concerning gene electrotransfer that has been accumulated over the last three decades. For that purpose, after briefly reviewing the medical applications that gene electrotransfer can provide, we outline membrane electropermeabilization, a key process for the delivery of DNA and smaller molecules. Since gene electrotransfer is a multipart process, we proceed our review in describing step by step our current understanding, with particular emphasis on DNA internalization and intracellular trafficking. Finally, we turn our attention to in vivo testing and methodology for gene electrotransfer. PMID:27029943

Assessment of the tumourigenic and metastatic properties of SK-MEL28 melanoma cells surviving electrochemotherapy with bleomycin

PubMed Central

Todorovic, Vesna; Sersa, Gregor; Mlakar, Vid; Glavac, Damjan; Cemazar, Maja

2012-01-01

Background Electrochemotherapy is a local treatment combining chemotherapy and electroporation and is highly effective treatment approach for subcutaneous tumours of various histologies. Contrary to surgery and radiation, the effect of electrochemotherapy on metastatic potential of tumour cells has not been extensively studied. The aim of the study was to evaluate the effect of electrochemotherapy with bleomycin on the metastatic potential of human melanoma cells in vitro. Materials and methods Viable cells 48 hours after electrochemotherapy were tested for their ability to migrate and invade through Matrigel coated porous membrane. In addition, microarray analysis and quantitative Real-Time PCR were used to detect changes in gene expression after electrochemotherapy. Results Cell migration and invasion were not changed in melanoma cells surviving electrochemotherapy. Interestingly, only a low number of tumourigenesis related genes was differentially expressed after electrochemotherapy. Conclusions Our data suggest that metastatic potential of human melanoma cells is not affected by electrochemotherapy with bleomycin, confirming safe role of electrochemotherapy in the clinics. PMID:22933978

Assessment of the tumourigenic and metastatic properties of SK-MEL28 melanoma cells surviving electrochemotherapy with bleomycin.

PubMed

Todorovic, Vesna; Sersa, Gregor; Mlakar, Vid; Glavac, Damjan; Cemazar, Maja

2012-03-01

Electrochemotherapy is a local treatment combining chemotherapy and electroporation and is highly effective treatment approach for subcutaneous tumours of various histologies. Contrary to surgery and radiation, the effect of electrochemotherapy on metastatic potential of tumour cells has not been extensively studied. The aim of the study was to evaluate the effect of electrochemotherapy with bleomycin on the metastatic potential of human melanoma cells in vitro. Viable cells 48 hours after electrochemotherapy were tested for their ability to migrate and invade through Matrigel coated porous membrane. In addition, microarray analysis and quantitative Real-Time PCR were used to detect changes in gene expression after electrochemotherapy. Cell migration and invasion were not changed in melanoma cells surviving electrochemotherapy. Interestingly, only a low number of tumourigenesis related genes was differentially expressed after electrochemotherapy. Our data suggest that metastatic potential of human melanoma cells is not affected by electrochemotherapy with bleomycin, confirming safe role of electrochemotherapy in the clinics.

Development of a High Temperature Microbial Fermentation Processfor Butanol Production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeor, Jeffery D.; Reed, David W.; Daubaras, Dayna L.

2016-06-01

Transforming renewable biomass into cost competitive high-performance biofuels and bioproducts is key to US energy security. Butanol production by microbial fermentation and chemical conversion to polyolefins, elastomers, drop-in jet or diesel fuel, and other chemicals is a promising solution. A high temperature fermentation process can facilitate butanol recovery up to 40%, by using gas stripping. Other benefits of fermentation at high temperatures are optimal hydrolysis rates in the saccharification of biomass which leads to maximized butanol production, decrease in energy costs associated with reactor cooling and capital cost associated with reactor design, and a decrease in contamination and cost formore » maintaining a sterile environment. Butanol stripping at elevated temperatures gives higher butanol production through constant removal and continuous fermentation. We describe methods used in an attempt to genetically prepare Geobacillus caldoxylosiliticus for insertion of a butanol pathway. Methods used were electroporation of electrocompetent cells, ternary conjugation with E. coli, and protoplast fusion.« less

Three-dimensional computational model of a blood oxygenator reconstructed from micro-CT scans.

PubMed

D'Onofrio, C; van Loon, R; Rolland, S; Johnston, R; North, L; Brown, S; Phillips, R; Sienz, J

2017-09-01

Cardiopulmonary bypass procedures are one of the most common operations and blood oxygenators are the centre piece for the heart-lung machines. Blood oxygenators have been tested as entire devices but intricate details on the flow field inside the oxygenators remain unknown. In this study, a novel method is presented to analyse the flow field inside oxygenators based on micro Computed Tomography (μCT) scans. Two Hollow Fibre Membrane (HFM) oxygenator prototypes were scanned and three-dimensional full scale models that capture the device-specific fibre distributions are set up for computational fluid dynamics analysis. The blood flow through the oxygenator is modelled as a non-Newtonian fluid. The results were compared against the flow solution through an ideal fibre distribution and show the importance of a uniform distribution of fibres and that the oxygenators analysed are not susceptible to flow directionality as mass flow versus area remain the same. However the pressure drop across the oxygenator is dependent on flow rate and direction. By comparing residence time of blood against the time frame to fully saturate blood with oxygen we highlight the potential of this method as design optimisation tool. In conclusion, image-based reconstruction is found to be a feasible route to assess oxygenator performance through flow modelling. It offers the possibility to review a product as manufactured rather than as designed, which is a valuable insight as a precursor to the approval processes. Finally, the flow analysis presented may be extended, at computational cost, to include species transport in further studies. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Plastic deformation treated as material flow through adjustable crystal lattice

NASA Astrophysics Data System (ADS)

Minakowski, P.; Hron, J.; Kratochvíl, J.; Kružík, M.; Málek, J.

2014-08-01

Looking at severe plastic deformation experiments, it seems that crystalline materials at yield behave as a special kind of anisotropic, highly viscous fluids flowing through an adjustable crystal lattice space. High viscosity provides a possibility to describe the flow as a quasi-static process, where inertial and other body forces can be neglected. The flow through the lattice space is restricted to preferred crystallographic planes and directions causing anisotropy. In the deformation process the lattice is strained and rotated. The proposed model is based on the rate form of the decomposition rule: the velocity gradient consists of the lattice velocity gradient and the sum of the velocity gradients corresponding to the slip rates of individual slip systems. The proposed crystal plasticity model allowing for large deformations is treated as the flow-adjusted boundary value problem. As a test example we analyze a plastic flow of an single crystal compressed in a channel die. We propose three step algorithm of finite element discretization for a numerical solution in the Arbitrary Lagrangian Eulerian (ALE) configuration.

Computation of incompressible viscous flows through artificial heart devices with moving boundaries

NASA Technical Reports Server (NTRS)

Kiris, Cetin; Rogers, Stuart; Kwak, Dochan; Chang, I.-DEE

1991-01-01

The extension of computational fluid dynamics techniques to artificial heart flow simulations is illustrated. Unsteady incompressible Navier-Stokes equations written in 3-D generalized curvilinear coordinates are solved iteratively at each physical time step until the incompressibility condition is satisfied. The solution method is based on the pseudo compressibility approach and uses an implicit upwind differencing scheme together with the Gauss-Seidel line relaxation method. The efficiency and robustness of the time accurate formulation of the algorithm are tested by computing the flow through model geometries. A channel flow with a moving indentation is computed and validated with experimental measurements and other numerical solutions. In order to handle the geometric complexity and the moving boundary problems, a zonal method and an overlapping grid embedding scheme are used, respectively. Steady state solutions for the flow through a tilting disk heart valve was compared against experimental measurements. Good agreement was obtained. The flow computation during the valve opening and closing is carried out to illustrate the moving boundary capability.

Microfluidic multiplexing of solid-state nanopores

NASA Astrophysics Data System (ADS)

Jain, Tarun; Rasera, Benjamin C.; Guerrero, Ricardo Jose S.; Lim, Jong-Min; Karnik, Rohit

2017-12-01

Although solid-state nanopores enable electronic analysis of many clinically and biologically relevant molecular structures, there are few existing device architectures that enable high-throughput measurement of solid-state nanopores. Herein, we report a method for microfluidic integration of multiple solid-state nanopores at a high density of one nanopore per (35 µm2). By configuring microfluidic devices with microfluidic valves, the nanopores can be rinsed from a single fluid input while retaining compatibility for multichannel electrical measurements. The microfluidic valves serve the dual purpose of fluidic switching and electric switching, enabling serial multiplexing of the eight nanopores with a single pair of electrodes. Furthermore, the device architecture exhibits low noise and is compatible with electroporation-based in situ nanopore fabrication, providing a scalable platform for automated electronic measurement of a large number of integrated solid-state nanopores.

Transient replication of BPV-1 requires two viral polypeptides encoded by the E1 and E2 open reading frames.

PubMed Central

Ustav, M; Stenlund, A

1991-01-01

Bovine papillomavirus (BPV) DNA is maintained as an episome with a constant copy number in transformed cells and is stably inherited. To study BPV replication we have developed a transient replication assay based on a highly efficient electroporation procedure. Using this assay we have determined that in the context of the viral genome two of the viral open reading frames, E1 and E2, are required for replication. Furthermore we show that when produced from expression vectors in the absence of other viral gene products, the full length E2 transactivator polypeptide and a 72 kd polypeptide encoded by the E1 open reading frame in its entirety, are both necessary and sufficient for replication BPV in C127 cells. Images PMID:1846806

Magnetic field effects for copper suspended nanofluid venture through a composite stenosed arteries with permeable wall

NASA Astrophysics Data System (ADS)

Akbar, Noreen Sher; Butt, Adil Wahid

2015-05-01

In the present paper magnetic field effects for copper nanoparticles for blood flow through composite stenosis in arteries with permeable wall are discussed. The copper nanoparticles for the blood flow with water as base fluid is not explored yet. The equations for the Cu-water nanofluid are developed first time in the literature and simplified using long wavelength and low Reynolds number assumptions. Exact solutions have been evaluated for velocity, pressure gradient, the solid volume fraction of the nanoparticles and temperature profile. The effect of various flow parameters on the flow and heat transfer characteristics is utilized.

Simultaneous injection-effective mixing analysis of palladium.

PubMed

Teshima, Norio; Noguchi, Daisuke; Joichi, Yasutaka; Lenghor, Narong; Ohno, Noriko; Sakai, Tadao; Motomizu, Shoji

2010-01-01

A novel concept of simultaneous injection-effective mixing analysis (SIEMA) is proposed, and a SIEMA method applied to the spectrophotometric determination of palladium using a water-soluble chromogenic reagent has been demonstrated. The flow configuration of SIEMA is a hybrid format of flow injection analysis (FIA), sequential injection analysis (SIA) and multicommutation in flow-based analysis. Sample and reagent solutions are aspirated into each holding coil through each solenoid valve by a syringe pump, and then the zones are simultaneously dispensed (injected) into a mixing coil by reversed flow toward a detector through a confluence point. This results in effective mixing and rapid detection with low reagent consumption.

Potatoes and Trout: Maintaining Robust Agriculture and a Healthy Trout Fishery in the Central Sands of Wisconsin

NASA Astrophysics Data System (ADS)

Fienen, M. N.; Bradbury, K. R.; Kniffin, M.; Barlow, P. M.; Krause, J.; Westenbroek, S.; Leaf, A.

2015-12-01

The well-drained sandy soil in the Wisconsin Central Sands is ideal for growing potatoes, corn, and other vegetables. A shallow sand and gravel aquifer provides abundant water for agricultural irrigation but also supplies critical base flow to cold-water trout streams. These needs compete with one another, and stakeholders from various perspectives are collaborating to seek solutions. Stakeholders were engaged in providing and verifying data to guide construction of a groundwater flow model which was used with linear and sequential linear programming to evaluate optimal tradeoffs between agricultural pumping and ecologically based minimum base flow values. The connection between individual irrigation wells as well as industrial and municipal supply and streamflow depletion can be evaluated using the model. Rather than addressing 1000s of wells individually, a variety of well management groups were established through k-means clustering. These groups are based on location, potential impact, water-use categories, depletion potential, and other factors. Through optimization, pumping rates were reduced to attain mandated minimum base flows. This formalization enables exploration of possible solutions for the stakeholders, and provides a tool which is transparent and forms a basis for discussion and negotiation.

40 CFR 1065.345 - Vacuum-side leak verification.

Code of Federal Regulations, 2011 CFR

2011-07-01

... zero flow, or by detecting the dilution of a known concentration of span gas when it flows through the.... Measure and record the absolute pressure of the trapped gas and optionally the system absolute temperature... pressure and optionally temperature. (4) Calculate the leak flow rate based on an assumed value of zero for...

40 CFR 1065.345 - Vacuum-side leak verification.

Code of Federal Regulations, 2014 CFR

2014-07-01

... zero flow, or by detecting the dilution of a known concentration of span gas when it flows through the.... Measure and record the absolute pressure of the trapped gas and optionally the system absolute temperature... pressure and optionally temperature. (4) Calculate the leak flow rate based on an assumed value of zero for...

40 CFR 1065.345 - Vacuum-side leak verification.

Code of Federal Regulations, 2013 CFR

2013-07-01

... zero flow, or by detecting the dilution of a known concentration of span gas when it flows through the.... Measure and record the absolute pressure of the trapped gas and optionally the system absolute temperature... pressure and optionally temperature. (4) Calculate the leak flow rate based on an assumed value of zero for...

40 CFR 1065.345 - Vacuum-side leak verification.

Code of Federal Regulations, 2010 CFR

2010-07-01

... zero flow, or by detecting the dilution of a known concentration of span gas when it flows through the.... Measure and record the absolute pressure of the trapped gas and optionally the system absolute temperature... pressure and optionally temperature. (4) Calculate the leak flow rate based on an assumed value of zero for...

40 CFR 1065.345 - Vacuum-side leak verification.

Code of Federal Regulations, 2012 CFR

2012-07-01

... zero flow, or by detecting the dilution of a known concentration of span gas when it flows through the.... Measure and record the absolute pressure of the trapped gas and optionally the system absolute temperature... pressure and optionally temperature. (4) Calculate the leak flow rate based on an assumed value of zero for...

Towards Natural Transition in Compressible Boundary Layers

DTIC Science & Technology

2016-06-29

Behaviour of a natural laminar flow aerofoil in flight through atmospheric turbulence. Journal of Fluid Mechanics, 767:394–429, 003 2015. [70] O...DISTRIBUTION STATEMENT A. Approved for public release; distribution is unlimited See report Wave packet, compressible boundary layer, subsonic flow ...Base flow generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 3.1.1 Boundary layer profiles

Development of an intradermal DNA vaccine delivery strategy to achieve single-dose immunity against respiratory syncytial virus.

PubMed

Smith, Trevor R F; Schultheis, Katherine; Morrow, Matthew P; Kraynyak, Kimberly A; McCoy, Jay R; Yim, Kevin C; Muthumani, Karuppiah; Humeau, Laurent; Weiner, David B; Sardesai, Niranjan Y; Broderick, Kate E

2017-05-15

Respiratory syncytial virus (RSV) is a massive medical burden in infants, children and the elderly worldwide, and an effective, safe RSV vaccine remains an unmet need. Here we assess a novel vaccination strategy based on the intradermal delivery of a SynCon® DNA-based vaccine encoding engineered RSV-F antigen using a surface electroporation device (SEP) to target epidermal cells, in clinically relevant experimental models. We demonstrate the ability of this strategy to elicit robust immune responses. Importantly we demonstrate complete resistance to pulmonary infection at a single low dose of vaccine in the cotton rat RSV/A challenge model. In contrast to the formalin-inactivated RSV (FI-RSV) vaccine, there was no enhanced lung inflammation upon virus challenge after DNA vaccination. In summary the data presented outline the pre-clinical development of a highly efficacious, tolerable and safe non-replicating vaccine delivery strategy. Copyright © 2017. Published by Elsevier Ltd.

Electrochemical cell operation and system

DOEpatents

Maru, Hansraj C.

1980-03-11

Thermal control in fuel cell operation is affected through sensible heat of process gas by providing common input manifolding of the cell gas flow passage in communication with the cell electrolyte and an additional gas flow passage which is isolated from the cell electrolyte and in thermal communication with a heat-generating surface of the cell. Flow level in the cell gas flow passage is selected based on desired output electrical energy and flow level in the additional gas flow passage is selected in accordance with desired cell operating temperature.

Performance Mapping Studies in Redox Flow Cells

NASA Technical Reports Server (NTRS)

Hoberecht, M. A.; Thaller, L. H.

1981-01-01

Pumping power requirements in any flow battery system constitute a direct parasitic energy loss. It is therefore useful to determine the practical lower limit for reactant flow rates. Through the use of a theoretical framework based on electrochemical first principles, two different experimental flow mapping techniques were developed to evaluate and compare electrodes as a function of flow rate. For the carbon felt electrodes presently used in NASA-Lewis Redox cells, a flow rate 1.5 times greater than the stoichiometric rate seems to be the required minimum.

Hydrothermal Circulation Within and Between Basement Outcrops on a Young Ridge Flank: Numerical Models and Thermal Constraints

NASA Astrophysics Data System (ADS)

Hutnak, M.; Fisher, A. T.; Stauffer, P.; Gable, C. W.

2005-12-01

We use two-dimensional, finite-element models of coupled heat and fluid flow to investigate local and large-scale heat and fluid transport around and between basement outcrops on a young ridge flank. System geometries and properties are based on observations and measurements on the 3.4-3.6 Ma eastern flank of the Juan de Fuca Ridge. A small area of basement exposure (Baby Bare outcrop) experiences focused hydrothermal discharge, whereas a much larger feature (Grizzly Bare outcrop) 50 km to the south is a site of hydrothermal recharge. Observations of seafloor heat flow, subseafloor pressures, and basement fluid geochemistry at and near these outcrops constrain acceptable model results. Single-outcrop simulations suggest that local convection alone (represented by a high Nusselt number proxy) cannot explain the near-outcrop heat flow patterns; rapid through-flow is required. Venting of at least 5 L/s through the smaller outcrop, a volumetric flow rate consistent with earlier estimates based on plume and outcrop measurements, is needed to match seafloor heat flow patterns. Heat flow patterns are more variable and complex near the larger, recharging outcrop. Simulations that include 5-20 L/s of recharge through this feature can replicate first-order trends in the data, but small-scale variations are likely to result from heterogeneous flow paths and vigorous, local convection. Two-outcrop simulations started with a warm hydrostatic initial condition, based on a conductive model, result in rapid fluid flow from the smaller outcrop to the larger outcrop, inconsistent with observations. Flow can be sustained in the opposite (correct) direction if it is initially forced, which generates a hydrothermal siphon between the two features. Free flow simulations maintain rapid circulation at rates consistent with observations (specific discharge of m/yr to tens of m/yr), provided basement permeability is on the order of 10-10 m2 or greater. Lateral flow rates scale inversely with the thickness of the permeable basement layer. The differential pressure needed to drive this circulation, created by the siphon, is on the order of tens to hundreds of kPa, with greater differential pressure needed when basement permeability is lower.

On the Symmetry of Molecular Flows Through the Pipe of an Arbitrary Shape (I) Diffusive Reflection

NASA Astrophysics Data System (ADS)

Kusumoto, Yoshiro

Molecular gas flows through the pipe of an arbitrary shape is mathematically considered based on a diffusive reflection model. To avoid a perpetual motion, the magnitude of the molecular flow rate must remain invariant under the exchange of inlet and outlet pressures. For this flow symmetry, the cosine law reflection at the pipe wall was found to be sufficient and necessary, on the assumption that the molecular flux is conserved in a collision with the wall. It was also shown that a spontaneous flow occurs in a hemispherical apparatus, if the reflection obeys the n-th power of cosine law with n other than unity. This apparatus could work as a molecular pump with no moving parts.

Casualty Handling Simulation Using the Scenario-based Engineering Process

DTIC Science & Technology

2000-02-28

HERPES ZOSTER ENCEPHALITIS HEPATITIS INFECTIOUS VIRAL ANIMAL BITES/RABIES EXPOSURE MUMPS INFECTIOUS MONONUCLEOSIS TRACHOMA STD-SYPHILIS STD...casualties as they flow through the system. Identifying the proper mix is complicated by many factors, including the specific casualty stream, the...casualties as they flow through the system. Identifying the proper mix is complicated by its dependence on mission types. For example, the types and

Laboratory Simulation of Flow through Single Fractured Granite

NASA Astrophysics Data System (ADS)

Singh, K. K.; Singh, D. N.; Ranjith, P. G.

2015-05-01

Laboratory simulation on fluid flow through fractured rock is important in addressing the seepage/fluid-in-rush related problems that occur during the execution of any civil or geological engineering projects. To understand the mechanics and transport properties of fluid through a fractured rock in detail and to quantify the sources of non-linearity in the discharge and base pressure relationship, fluid flow experiments were carried out on a cylindrical sample of granite containing a `single rough walled fracture'. These experiments were performed under varied conditions of confining pressures, σ 3 (5-40 MPa), which can simulate the condition occurring about 1,000 m below in the earth crust, with elevated base pressure, b p (up to 25 MPa) and by changing fracture roughness. The details of the methodologies involved and the observations are discussed here. The obtained results indicate that most of the data in the Q verses b p plot, fall on the straight line and the flow through the single fracture in granite obeys Darcy's law or the well-known "cubic law" even at high value of b p (=4 MPa) and σ 3 (=5 MPa) combination. The Reynolds number is quite sensitive to the b p, σ 3 and fracture roughness, and there is a critical b p, beyond which transition in flow occurs from laminar to turbulent. It is believed that such studies will be quite useful in identifying the limits of applicability of well know `cubic law', which is required for precise calculation of discharge and/or aperture in any practical issues and in further improving theoretical/numerical models associated with fluid flow through a single fracture.

The Effect of Millisecond Pulsed Electric Fields (msPEF) on Intracellular Drug Transport with Negatively Charged Large Nanocarriers Made of Solid Lipid Nanoparticles (SLN): In Vitro Study.

PubMed

Kulbacka, Julita; Pucek, Agata; Wilk, Kazimiera Anna; Dubińska-Magiera, Magda; Rossowska, Joanna; Kulbacki, Marek; Kotulska, Małgorzata

2016-10-01

Drug delivery technology is still a dynamically developing field of medicine. The main direction in nanotechnology research (nanocarriers, nanovehicles, etc.) is efficient drug delivery to target cells with simultaneous drug reduction concentration. However, nanotechnology trends in reducing the carrier sizes to several nanometers limit the volume of the loaded substance and may pose a danger of uncontrolled access into the cells. On the other hand, nanoparticles larger than 200 nm in diameter have difficulties to undergo rapid diffusional transport through cell membranes. The main advantage of large nanoparticles is higher drug encapsulation efficiency and the ability to deliver a wider array of drugs. Our present study contributes a new approach with large Tween 80 solid lipid nanoparticles SLN (i.e., hydrodynamic GM-SLN-glycerol monostearate, GM, as the lipid and ATO5-SLNs-glyceryl palmitostearate, ATO5, as the lipid) with diameters DH of 379.4 nm and 547 nm, respectively. They are used as drug carriers alone and in combination with electroporation (EP) induced by millisecond pulsed electric fields. We evaluate if EP can support the transport of large nanocarriers into cells. The study was performed with two cell lines: human colon adenocarcinoma LoVo and hamster ovarian fibroblastoid CHO-K1 with coumarin 6 (C6) as a fluorescent marker for encapsulation. The biological safety of the potential treatment procedure was evaluated with cell viability after their exposure to nanoparticles and EP. The EP efficacy was evaluated by FACS method. The impact on intracellular structure organization of cytoskeleton was visualized by CLSM method with alpha-actin and beta-tubulin. The obtained results indicate low cytotoxicity of both carrier types, free and loaded with C6. The evaluation of cytoskeleton proteins indicated no intracellular structure damage. The intracellular uptake and accumulation show that SLNs do not support transport of C6 coumarin. Only application of electroporation improved the transport of encapsulated and free C6 into both treated cell lines.

Flows of bottom water in fractures of the North Mid-Atlantic Ridge

NASA Astrophysics Data System (ADS)

Morozov, E. G.; Tarakanov, R. Yu.; Demidova, T. A.; Makarenko, N. I.

2017-06-01

It has been shown that the total transport of Antarctic Bottom Water (AABW) in the northern fractures (Kane, Cabo Verde, Marathon) are one order of magnitude smaller than in the southern fractures (Vema, Doldrums, Vernadsky). The estimates of AABW transport through this group of fractures based on measurements in 2014 were approximately 0.28 Sv, which is about 25% of the transport through the Vema Fracture Zone. However, the coldest water flows through the Vema Fracture Zone.

Robustness analysis of complex networks with power decentralization strategy via flow-sensitive centrality against cascading failures

NASA Astrophysics Data System (ADS)

Guo, Wenzhang; Wang, Hao; Wu, Zhengping

2018-03-01

Most existing cascading failure mitigation strategy of power grids based on complex network ignores the impact of electrical characteristics on dynamic performance. In this paper, the robustness of the power grid under a power decentralization strategy is analysed through cascading failure simulation based on AC flow theory. The flow-sensitive (FS) centrality is introduced by integrating topological features and electrical properties to help determine the siting of the generation nodes. The simulation results of the IEEE-bus systems show that the flow-sensitive centrality method is a more stable and accurate approach and can enhance the robustness of the network remarkably. Through the study of the optimal flow-sensitive centrality selection for different networks, we find that the robustness of the network with obvious small-world effect depends more on contribution of the generation nodes detected by community structure, otherwise, contribution of the generation nodes with important influence on power flow is more critical. In addition, community structure plays a significant role in balancing the power flow distribution and further slowing the propagation of failures. These results are useful in power grid planning and cascading failure prevention.

Aptamer-fluorescent silica nanoparticles bioconjugates based dual-color flow cytometry for specific detection of Staphylococcus aureus.

PubMed

He, Xiaoxiao; Li, Yuhong; He, Dinggen; Wang, Kemin; Shangguan, Jingfang; Shi, Hui

2014-07-01

This paper describes a sensitive and specific determination strategy for Staphylococcus aureus (S. aureus) detection using aptamer recognition and fluorescent silica nanoparticles (FSiNPs) label based dual-color flow cytometry assay (Aptamer/FSiNPs-DCFCM). In the protocol, an aptamer, having high affinity to S. aureus, was first covalently immobilized onto chloropropyl functionalized FSiNPs through a click chemistry approach to generate aptamer-nanoparticles bioconjugates (Aptamer/FSiNPs). Next, S. aureus was incubated with Aptamer/FSiNPs, and then stained with SYBR Green I (a special staining material for the duplex DNA). Upon target binding and nucleic acid staining with SYBR Green I, the S. aureus was determined using two-color flow cytometry. The method took advantage of the specificity of aptamer, signal amplification of FSiNPs label and decreased false positives of two-color flow cytometry assay. It was demonstrated that these Aptamer/FSiNPs could efficiently recognize and fluorescently label target S. aureus. Through multiparameter determination with flow cytometry, this assay allowed for detection of as low as 1.5 x 10(2) and 7.6 x 10(2) cells mL(-1) S. aureus in buffer and spiked milk, respectively, with higher sensitivity than the Aptamer/FITC based flow cytometry.

Free surface profiles in river flows: Can standard energy-based gradually-varied flow computations be pursued?

NASA Astrophysics Data System (ADS)

Cantero, Francisco; Castro-Orgaz, Oscar; Garcia-Marín, Amanda; Ayuso, José Luis; Dey, Subhasish

2015-10-01

Is the energy equation for gradually-varied flow the best approximation for the free surface profile computations in river flows? Determination of flood inundation in rivers and natural waterways is based on the hydraulic computation of flow profiles. This is usually done using energy-based gradually-varied flow models, like HEC-RAS, that adopts a vertical division method for discharge prediction in compound channel sections. However, this discharge prediction method is not so accurate in the context of advancements over the last three decades. This paper firstly presents a study of the impact of discharge prediction on the gradually-varied flow computations by comparing thirteen different methods for compound channels, where both energy and momentum equations are applied. The discharge, velocity distribution coefficients, specific energy, momentum and flow profiles are determined. After the study of gradually-varied flow predictions, a new theory is developed to produce higher-order energy and momentum equations for rapidly-varied flow in compound channels. These generalized equations enable to describe the flow profiles with more generality than the gradually-varied flow computations. As an outcome, results of gradually-varied flow provide realistic conclusions for computations of flow in compound channels, showing that momentum-based models are in general more accurate; whereas the new theory developed for rapidly-varied flow opens a new research direction, so far not investigated in flows through compound channels.

In vivo protection against ZIKV infection and pathogenesis through passive antibody transfer and active immunisation with a prMEnv DNA vaccine.

PubMed

Muthumani, Karuppiah; Griffin, Bryan D; Agarwal, Sangya; Kudchodkar, Sagar B; Reuschel, Emma L; Choi, Hyeree; Kraynyak, Kimberly A; Duperret, Elizabeth K; Keaton, Amelia Anne; Chung, Christopher; Kim, Yinho K; Booth, Stephanie A; Racine, Trina; Yan, Jian; Morrow, Matthew P; Jiang, Jingjing; Lee, Brian; Ramos, Stephanie; Broderick, Kate E; Reed, Charles C; Khan, Amir S; Humeau, Laurent; Ugen, Kenneth E; Park, Young K; Maslow, Joel N; Sardesai, Niranjan Y; Joseph Kim, J; Kobinger, Gary P; Weiner, David B

2016-01-01

Significant concerns have been raised owing to the rapid global spread of infection and disease caused by the mosquito-borne Zika virus (ZIKV). Recent studies suggest that ZIKV can also be transmitted sexually, further increasing the exposure risk for this virus. Associated with this spread is a dramatic increase in cases of microcephaly and additional congenital abnormalities in infants of ZIKV-infected mothers, as well as a rise in the occurrence of Guillain Barre' syndrome in infected adults. Importantly, there are no licensed therapies or vaccines against ZIKV infection. In this study, we generate and evaluate the in vivo efficacy of a novel, synthetic, DNA vaccine targeting the pre-membrane+envelope proteins (prME) of ZIKV. Following initial in vitro development and evaluation studies of the plasmid construct, mice and non-human primates were immunised with this prME DNA-based immunogen through electroporation-mediated enhanced DNA delivery. Vaccinated animals were found to generate antigen-specific cellular and humoral immunity and neutralisation activity. In mice lacking receptors for interferon (IFN)-α/β (designated IFNAR -/- ) immunisation with this DNA vaccine induced, following in vivo viral challenge, 100% protection against infection-associated weight loss or death in addition to preventing viral pathology in brain tissue. In addition, passive transfer of non-human primate anti-ZIKV immune serum protected IFNAR -/- mice against subsequent viral challenge. This study in NHP and in a pathogenic mouse model supports the importance of immune responses targeting prME in ZIKV infection and suggests that additional research on this vaccine approach may have relevance for ZIKV control and disease prevention in humans.

In vivo protection against ZIKV infection and pathogenesis through passive antibody transfer and active immunisation with a prMEnv DNA vaccine

PubMed Central

Muthumani, Karuppiah; Griffin, Bryan D; Agarwal, Sangya; Kudchodkar, Sagar B; Reuschel, Emma L; Choi, Hyeree; Kraynyak, Kimberly A; Duperret, Elizabeth K; Keaton, Amelia Anne; Chung, Christopher; Kim, Yinho K; Booth, Stephanie A; Racine, Trina; Yan, Jian; Morrow, Matthew P; Jiang, Jingjing; Lee, Brian; Ramos, Stephanie; Broderick, Kate E; Reed, Charles C; Khan, Amir S; Humeau, Laurent; Ugen, Kenneth E; Park, Young K; Maslow, Joel N; Sardesai, Niranjan Y; Joseph Kim, J; Kobinger, Gary P; Weiner, David B

2016-01-01

Significant concerns have been raised owing to the rapid global spread of infection and disease caused by the mosquito-borne Zika virus (ZIKV). Recent studies suggest that ZIKV can also be transmitted sexually, further increasing the exposure risk for this virus. Associated with this spread is a dramatic increase in cases of microcephaly and additional congenital abnormalities in infants of ZIKV-infected mothers, as well as a rise in the occurrence of Guillain Barre’ syndrome in infected adults. Importantly, there are no licensed therapies or vaccines against ZIKV infection. In this study, we generate and evaluate the in vivo efficacy of a novel, synthetic, DNA vaccine targeting the pre-membrane+envelope proteins (prME) of ZIKV. Following initial in vitro development and evaluation studies of the plasmid construct, mice and non-human primates were immunised with this prME DNA-based immunogen through electroporation-mediated enhanced DNA delivery. Vaccinated animals were found to generate antigen-specific cellular and humoral immunity and neutralisation activity. In mice lacking receptors for interferon (IFN)-α/β (designated IFNAR−/−) immunisation with this DNA vaccine induced, following in vivo viral challenge, 100% protection against infection-associated weight loss or death in addition to preventing viral pathology in brain tissue. In addition, passive transfer of non-human primate anti-ZIKV immune serum protected IFNAR−/− mice against subsequent viral challenge. This study in NHP and in a pathogenic mouse model supports the importance of immune responses targeting prME in ZIKV infection and suggests that additional research on this vaccine approach may have relevance for ZIKV control and disease prevention in humans. PMID:29263859

MyD88/CD40 Genetic Adjuvant Function in Cutaneous Atypical Antigen-Presenting Cells Contributes to DNA Vaccine Immunogenicity

PubMed Central

Slawin, Kevin M.; Levitt, Jonathan M.; Spencer, David M.

2016-01-01

Therapeutic DNA-based vaccines aim to prime an adaptive host immune response against tumor-associated antigens, eliminating cancer cells primarily through CD8+ cytotoxic T cell-mediated destruction. To be optimally effective, immunological adjuvants are required for the activation of tumor-specific CD8+ T cells responses by DNA vaccination. Here, we describe enhanced anti-tumor efficacy of an in vivo electroporation-delivered DNA vaccine by inclusion of a genetically encoded chimeric MyD88/CD40 (MC) adjuvant, which integrates both innate and adaptive immune signaling pathways. When incorporated into a DNA vaccine, signaling by the MC adjuvant increased antigen-specific CD8+ T cells and promoted elimination of pre-established tumors. Interestingly, MC-enhanced vaccine efficacy did not require direct-expression of either antigen or adjuvant by local antigen-presenting cells, but rather our data supports a key role for MC function in “atypical” antigen-presenting cells of skin. In particular, MC adjuvant-modified keratinocytes increased inflammatory cytokine secretion, upregulated surface MHC class I, and were able to increase in vitro and in vivo priming of antigen-specific CD8+ T cells. Furthermore, in the absence of critical CD8α+/CD103+ cross-priming dendritic cells, MC was still able to promote immune priming in vivo, albeit at a reduced level. Altogether, our data support a mechanism by which MC signaling activates an inflammatory phenotype in atypical antigen-presenting cells within the cutaneous vaccination site, leading to an enhanced CD8+ T cell response against DNA vaccine-encoded antigens, through both CD8α+/CD103+ dendritic cell-dependent and independent pathways. PMID:27741278

Fracture control of ground water flow and water chemistry in a rock aquitard.

PubMed

Eaton, Timothy T; Anderson, Mary P; Bradbury, Kenneth R

2007-01-01

There are few studies on the hydrogeology of sedimentary rock aquitards although they are important controls in regional ground water flow systems. We formulate and test a three-dimensional (3D) conceptual model of ground water flow and hydrochemistry in a fractured sedimentary rock aquitard to show that flow dynamics within the aquitard are more complex than previously believed. Similar conceptual models, based on regional observations and recently emerging principles of mechanical stratigraphy in heterogeneous sedimentary rocks, have previously been applied only to aquifers, but we show that they are potentially applicable to aquitards. The major elements of this conceptual model, which is based on detailed information from two sites in the Maquoketa Formation in southeastern Wisconsin, include orders of magnitude contrast between hydraulic diffusivity (K/S(s)) of fractured zones and relatively intact aquitard rock matrix, laterally extensive bedding-plane fracture zones extending over distances of over 10 km, very low vertical hydraulic conductivity of thick shale-rich intervals of the aquitard, and a vertical hydraulic head profile controlled by a lateral boundary at the aquitard subcrop, where numerous surface water bodies dominate the shallow aquifer system. Results from a 3D numerical flow model based on this conceptual model are consistent with field observations, which did not fit the typical conceptual model of strictly vertical flow through an aquitard. The 3D flow through an aquitard has implications for predicting ground water flow and for planning and protecting water supplies.

Fracture control of ground water flow and water chemistry in a rock aquitard

USGS Publications Warehouse

Eaton, T.T.; Anderson, M.P.; Bradbury, K.R.

2007-01-01

There are few studies on the hydrogeology of sedimentary rock aquitards although they are important controls in regional ground water flow systems. We formulate and test a three-dimensional (3D) conceptual model of ground water flow and hydrochemistry in a fractured sedimentary rock aquitard to show that flow dynamics within the aquitard are more complex than previously believed. Similar conceptual models, based on regional observations and recently emerging principles of mechanical stratigraphy in heterogeneous sedimentary rocks, have previously been applied only to aquifers, but we show that they are potentially applicable to aquitards. The major elements of this conceptual model, which is based on detailed information from two sites in the Maquoketa Formation in southeastern Wisconsin, include orders of magnitude contrast between hydraulic diffusivity (K/Ss) of fractured zones and relatively intact aquitard rock matrix, laterally extensive bedding-plane fracture zones extending over distances of over 10 km, very low vertical hydraulic conductivity of thick shale-rich intervals of the aquitard, and a vertical hydraulic head profile controlled by a lateral boundary at the aquitard subcrop, where numerous surface water bodies dominate the shallow aquifer system. Results from a 3D numerical flow model based on this conceptual model are consistent with field observations, which did not fit the typical conceptual model of strictly vertical flow through an aquitard. The 3D flow through an aquitard has implications for predicting ground water flow and for planning and protecting water supplies. ?? 2007 National Ground Water Association.

Modeling of Ice Flow and Internal Layers Along a Flow Line Through Swiss Camp in West Greenland

NASA Technical Reports Server (NTRS)

Wang, W. L.; Zwally, H. Jay; Abdalati, W.; Luo, S.; Koblinsky, Chester J. (Technical Monitor)

2001-01-01

An anisotropic ice flow line model is applied to a flow line through Swiss Camp (69.57 N, 49.28 W) in West Greenland to estimate the dates of internal layers detected by Radio-Echo Sounding measurements. The effect of an anisotropic ice fabric on ice flow is incorporated into the steady state flow line model. The stress-strain rate relationship for anisotropic ice is characterized by an enhancement factor based on the laboratory observations of ice deformation under combined compression and shear stresses. By using present-day data of accumulation rate, surface temperature, surface elevation and ice thickness along the flow line as model inputs, a very close agreement is found between the isochrones generated from the model and the observed internal layers with confirmed dates. The results indicate that this part of Greenland ice sheet is primarily in steady state.

Hydrodynamic bifurcation in electro-osmotically driven periodic flows

NASA Astrophysics Data System (ADS)

Morozov, Alexander; Marenduzzo, Davide; Larson, Ronald G.

2018-06-01

In this paper, we report an inertial instability that occurs in electro-osmotically driven channel flows. We assume that the charge motion under the influence of an externally applied electric field is confined to a small vicinity of the channel walls that, effectively, drives a bulk flow through a prescribed slip velocity at the boundaries. Here, we study spatially periodic wall velocity modulations in a two-dimensional straight channel numerically. At low slip velocities, the bulk flow consists of a set of vortices along each wall that are left-right symmetric, while at sufficiently high slip velocities, this flow loses its stability through a supercritical bifurcation. Surprisingly, the flow state that bifurcates from a left-right symmetric base flow has a rather strong mean component along the channel, which is similar to pressure-driven velocity profiles. The instability sets in at rather small Reynolds numbers of about 20-30, and we discuss its potential applications in microfluidic devices.

Exhaust gas bypass valve control for thermoelectric generator

DOEpatents

Reynolds, Michael G; Yang, Jihui; Meisner, Greogry P.; Stabler, Francis R.; De Bock, Hendrik Pieter Jacobus; Anderson, Todd Alan

2012-09-04

A method of controlling engine exhaust flow through at least one of an exhaust bypass and a thermoelectric device via a bypass valve is provided. The method includes: determining a mass flow of exhaust exiting an engine; determining a desired exhaust pressure based on the mass flow of exhaust; comparing the desired exhaust pressure to a determined exhaust pressure; and determining a bypass valve control value based on the comparing, wherein the bypass valve control value is used to control the bypass valve.

A workstation based simulator for teaching compressible aerodynamics

NASA Technical Reports Server (NTRS)

Benson, Thomas J.

1994-01-01

A workstation-based interactive flow simulator has been developed to aid in the teaching of undergraduate compressible aerodynamics. By solving the equations found in NACA 1135, the simulator models three basic fluids problems encountered in supersonic flow: flow past a compression corner, flow past two wedges in series, and flow past two opposed wedges. The study can vary the geometry or flow conditions through a graphical user interface and the new conditions are calculated immediately. Various graphical formats present the results of the flow calculations to the student. The simulator includes interactive questions and answers to aid in both the use of the tool and to develop an understanding of some of the complexities of compressible aerodynamics. A series of help screens make the simulator easy to learn and use.

Reliable multicast protocol specifications flow control and NACK policy

NASA Technical Reports Server (NTRS)

Callahan, John R.; Montgomery, Todd L.; Whetten, Brian

1995-01-01

This appendix presents the flow and congestion control schemes recommended for RMP and a NACK policy based on the whiteboard tool. Because RMP uses a primarily NACK based error detection scheme, there is no direct feedback path through which receivers can signal losses through low buffer space or congestion. Reliable multicast protocols also suffer from the fact that throughput for a multicast group must be divided among the members of the group. This division is usually very dynamic in nature and therefore does not lend itself well to a priori determination. These facts have led the flow and congestion control schemes of RMP to be made completely orthogonal to the protocol specification. This allows several differing schemes to be used in different environments to produce the best results. As a default, a modified sliding window scheme based on previous algorithms are suggested and described below.

Unsteady Analysis of Turbine Main Flow Coupled with Secondary Air Flow

NASA Technical Reports Server (NTRS)

Hah, Chunill

2006-01-01

Two numerical approaches are used to model the interaction between the turbine main gas flow and the wheelspace cavity seal flow. The 3-D, unsteady Reynolds-averaged Navier-Stokes equations are solved with a CFD code based on a structured grid to study the interaction between the turbine main gas flow and the wheelspace cavity seal flow. A CFD code based on an unstructured grid is used to solve detailed flow feature in the cavity seal which has a complex geometry. The numerical results confirm various observations from earlier experimental studies under similar flow conditions. When the flow rate through the rim cavity seal is increased, the ingestion of the main turbine flow into the rim seal area decreases drastically. However, a small amount of main gas flow is ingested to the rim seal area even with very high level of seal flow rate. This is due to the complex nature of 3-D, unsteady flow interaction near the hub of the turbine stage.

Examining Passenger Flow Choke Points at Airports Using Discrete Event Simulation

NASA Technical Reports Server (NTRS)

Brown, Jeremy R.; Madhavan, Poomima

2011-01-01

The movement of passengers through an airport quickly, safely, and efficiently is the main function of the various checkpoints (check-in, security. etc) found in airports. Human error combined with other breakdowns in the complex system of the airport can disrupt passenger flow through the airport leading to lengthy waiting times, missing luggage and missed flights. In this paper we present a model of passenger flow through an airport using discrete event simulation that will provide a closer look into the possible reasons for breakdowns and their implications for passenger flow. The simulation is based on data collected at Norfolk International Airport (ORF). The primary goal of this simulation is to present ways to optimize the work force to keep passenger flow smooth even during peak travel times and for emergency preparedness at ORF in case of adverse events. In this simulation we ran three different scenarios: real world, increased check-in stations, and multiple waiting lines. Increased check-in stations increased waiting time and instantaneous utilization. while the multiple waiting lines decreased both the waiting time and instantaneous utilization. This simulation was able to show how different changes affected the passenger flow through the airport.

A Unique Opportunity to Test Whether Cell Fusion is a Mechanism of Breast Cancer Metastasis

DTIC Science & Technology

2013-07-01

populations. Last cycle we optimized electroporation conditions for T47D and human mesenchymal stem cell populations and this cycle we have improved our...specific receptor-ligand interactions necessary for cell fusion, to produce a target for drug therapy. Post-fusion events might also be investigated...new tools for the study of the complex processes of cell fusion. The inducible bipartite nature of these strategies assures the accurate

Preventing Bandwidth Abuse at the Router through Sending Rate Estimate-based Active Queue Management

DTIC Science & Technology

2007-06-01

behavior is growing in the Internet. These non-responsive sources can monopolize network bandwidth and starve the “congestion friendly” flows. Without...unnecessarily complex because most of the flows in the Internet are short flows usually termed as “web mice ” [7]. Moreover, having a separate queue for each

The role of flow in the morphodynamics of embryonic heart

NASA Astrophysics Data System (ADS)

Gharib, Morteza

2017-11-01

Nature has shown us that some hearts do not require valves to achieve unidirectional flow. In its earliest stages, the vertebrate heart consists of a primitive tube that drives blood through a simple vascular network nourishing tissues and other developing organ systems. We have shown that in the case of the embryonic zebrafish heart, an elastic wave resonance mechanism based on impedance mismatches at the boundaries of the heart tube is the likely mechanism responsible for the valveless pumping behavior. When functioning normally, mature heart valves prevent intracardiac retrograde blood flow; before valves develop there is considerable regurgitation, resulting in oscillatory flow between the atrium and ventricle. We show that reversing flows are particularly strong stimuli to endothelial cells and that heart valves form as a developmental response to oscillatory blood flow through the maturing heart.

Cells exposed to nanosecond electrical pulses exhibit biomarkers of mechanical stress

NASA Astrophysics Data System (ADS)

Roth, Caleb C.; Barnes, Ronald A.; Ibey, Bennett L.; Beier, Hope T.; Moen, Erick K.; Glickman, Randolph D.

2015-03-01

Exposure of cells to very short (<1 μs) electric pulses in the megavolt/meter range have been shown to cause disruption of the plasma membrane. This disruption is often characterized by the formation of numerous small pores (<2 nm in diameter) in the plasma membrane that last for several minutes, allowing the flow of ions into the cell. These small pores are called nanopores and the resulting damage to the plasma membrane is referred to as nanoporation. Nanosecond electrical pulse (nsEP) exposure can impart many different stressors on a cell, including electrical, electro-chemical, and mechanical stress. Thus, nsEP exposure is not a "clean" insult, making determination of the mechanism of nanoporation quite difficult. We hypothesize that nsEP exposure creates acoustic shock waves capable of causing nanoporation. Microarray analysis of primary adult human dermal fibroblasts (HDFa) exposed to nsEP, indicated several genes associated with mechanical stress were selectively upregulated 4 h post exposure. The idea that nanoporation is caused by external mechanical force from acoustic shock waves has, to our knowledge, not been investigated. This work will critically challenge the existing paradigm that nanoporation is caused solely by an electric-field driven event and could provide the basis for a plausible explanation for electroporation.

A knowledge-based approach to automated flow-field zoning for computational fluid dynamics

NASA Technical Reports Server (NTRS)

Vogel, Alison Andrews

1989-01-01

An automated three-dimensional zonal grid generation capability for computational fluid dynamics is shown through the development of a demonstration computer program capable of automatically zoning the flow field of representative two-dimensional (2-D) aerodynamic configurations. The applicability of a knowledge-based programming approach to the domain of flow-field zoning is examined. Several aspects of flow-field zoning make the application of knowledge-based techniques challenging: the need for perceptual information, the role of individual bias in the design and evaluation of zonings, and the fact that the zoning process is modeled as a constructive, design-type task (for which there are relatively few examples of successful knowledge-based systems in any domain). Engineering solutions to the problems arising from these aspects are developed, and a demonstration system is implemented which can design, generate, and output flow-field zonings for representative 2-D aerodynamic configurations.

Targeted and controlled anticancer drug delivery and release with magnetoelectric nanoparticles

NASA Astrophysics Data System (ADS)

Rodzinski, Alexandra; Guduru, Rakesh; Liang, Ping; Hadjikhani, Ali; Stewart, Tiffanie; Stimphil, Emmanuel; Runowicz, Carolyn; Cote, Richard; Altman, Norman; Datar, Ram; Khizroev, Sakhrat

2016-02-01

It is a challenge to eradicate tumor cells while sparing normal cells. We used magnetoelectric nanoparticles (MENs) to control drug delivery and release. The physics is due to electric-field interactions (i) between MENs and a drug and (ii) between drug-loaded MENs and cells. MENs distinguish cancer cells from normal cells through the membrane’s electric properties; cancer cells have a significantly smaller threshold field to induce electroporation. In vitro and in vivo studies (nude mice with SKOV-3 xenografts) showed that (i) drug (paclitaxel (PTX)) could be attached to MENs (30-nm CoFe2O4@BaTiO3 nanostructures) through surface functionalization to avoid its premature release, (ii) drug-loaded MENs could be delivered into cancer cells via application of a d.c. field (~100 Oe), and (iii) the drug could be released off MENs on demand via application of an a.c. field (~50 Oe, 100 Hz). The cell lysate content was measured with scanning probe microscopy and spectrophotometry. MENs and control ferromagnetic and polymer nanoparticles conjugated with HER2-neu antibodies, all loaded with PTX were weekly administrated intravenously. Only the mice treated with PTX-loaded MENs (15/200 μg) in a field for three months were completely cured, as confirmed through infrared imaging and post-euthanasia histology studies via energy-dispersive spectroscopy and immunohistochemistry.

Predicting commuter flows in spatial networks using a radiation model based on temporal ranges

NASA Astrophysics Data System (ADS)

Ren, Yihui; Ercsey-Ravasz, Mária; Wang, Pu; González, Marta C.; Toroczkai, Zoltán

2014-11-01

Understanding network flows such as commuter traffic in large transportation networks is an ongoing challenge due to the complex nature of the transportation infrastructure and human mobility. Here we show a first-principles based method for traffic prediction using a cost-based generalization of the radiation model for human mobility, coupled with a cost-minimizing algorithm for efficient distribution of the mobility fluxes through the network. Using US census and highway traffic data, we show that traffic can efficiently and accurately be computed from a range-limited, network betweenness type calculation. The model based on travel time costs captures the log-normal distribution of the traffic and attains a high Pearson correlation coefficient (0.75) when compared with real traffic. Because of its principled nature, this method can inform many applications related to human mobility driven flows in spatial networks, ranging from transportation, through urban planning to mitigation of the effects of catastrophic events.

Environmental continuous air monitor inlet with combined preseparator and virtual impactor

DOEpatents

Rodgers, John C [Santa Fe, NM

2007-06-19

An inlet for an environmental air monitor is described wherein a pre-separator interfaces with ambient environment air and removes debris and insects commonly associated with high wind outdoors and a deflector plate in communication with incoming air from the pre-separator stage, that directs the air radially and downward uniformly into a plurality of accelerator jets located in a manifold of a virtual impactor, the manifold being cylindrical and having a top, a base, and a wall, with the plurality of accelerator jets being located in the top of the manifold and receiving the directed air and accelerating directed air, thereby creating jets of air penetrating into the manifold, where a major flow is deflected to the walls of the manifold and extracted through ports in the walls. A plurality of receiver nozzles are located in the base of the manifold coaxial with the accelerator jets, and a plurality of matching flow restrictor elements are located in the plurality of receiver nozzles for balancing and equalizing the total minor flow among all the plurality of receiver nozzles, through which a lower, fractional flow extracts large particle constituents of the air for collection on a sample filter after passing through the plurality of receiver nozzles and the plurality of matching flow restrictor elements.

AC Electroosmotic Pumping in Nanofluidic Funnels.

PubMed

Kneller, Andrew R; Haywood, Daniel G; Jacobson, Stephen C

2016-06-21

We report efficient pumping of fluids through nanofluidic funnels when a symmetric AC waveform is applied. The asymmetric geometry of the nanofluidic funnel induces not only ion current rectification but also electroosmotic flow rectification. In the base-to-tip direction, the funnel exhibits a lower ion conductance and a higher electroosmotic flow velocity, whereas, in the tip-to-base direction, the funnel has a higher ion conductance and a lower electroosmotic flow velocity. Consequently, symmetric AC waveforms easily pump fluid through the nanofunnels over a range of frequencies, e.g., 5 Hz to 5 kHz. In our experiments, the nanofunnels were milled into glass substrates with a focused ion beam (FIB) instrument, and the funnel design had a constant 5° taper with aspect ratios (funnel tip width to funnel depth) of 0.1 to 1.0. We tracked ion current rectification by current-voltage (I-V) response and electroosmotic flow rectification by transport of a zwitterionic fluorescent probe. Rectification of ion current and electroosmotic flow increased with increasing electric field applied to the nanofunnel. Our results support three-dimensional simulations of ion transport and electroosmotic transport through nanofunnels, which suggest the asymmetric electroosmotic transport stems from an induced pressure at the junction of the nanochannel and nanofunnel tip.

Fiber optic liquid mass flow sensor and method

NASA Technical Reports Server (NTRS)

Korman, Valentin (Inventor); Gregory, Don Allen (Inventor); Wiley, John T. (Inventor); Pedersen, Kevin W. (Inventor)

2010-01-01

A method and apparatus are provided for sensing the mass flow rate of a fluid flowing through a pipe. A light beam containing plural individual wavelengths is projected from one side of the pipe across the width of the pipe so as to pass through the fluid under test. Fiber optic couplers located at least two positions on the opposite side of the pipe are used to detect the light beam. A determination is then made of the relative strengths of the light beam for each wavelength at the at least two positions and based at least in part on these relative strengths, the mass flow rate of the fluid is determined.