Tracking Lysosome Migration within Chinese Hamster Ovary (CHO) Cells Following Exposure to Nanosecond Pulsed Electric Fields

Above a threshold electric field strength, 600 ns-duration pulsed electric field (nsPEF) exposure substantially porates and permeabilizes cellular plasma membranes in aqueous solution to many small ions. Repetitive exposures increase permeabilization to calcium ions (Ca2+) in a dosage-dependent manner. Such exposure conditions can create relatively long-lived pores that reseal after passive lateral diffusion of lipids should have closed the pores. One explanation for eventual pore resealing is active membrane repair, and an ubiquitous repair mechanism in mammalian cells is lysosome exocytosis. A previous study shows that intracellular lysosome movement halts upon a 16.2 kV/cm, 600-ns PEF exposure of a single train of 20 pulses at 5 Hz. In that study, lysosome stagnation qualitatively correlates with the presence of Ca2+ in the extracellular solution and with microtubule collapse. The present study tests the hypothesis that limitation of nsPEF-induced Ca2+ influx and colloid osmotic cell swelling permits unabated lysosome translocation in exposed cells. The results indicate that the efforts used herein to preclude Ca2+ influx and colloid osmotic swelling following nsPEF exposure did not prevent attenuation of lysosome translocation. Intracellular lysosome movement is inhibited by nsPEF exposure(s) in the presence of PEG 300-containing solution or by 20 pulses of nsPEF in the presence of extracellular calcium. The only cases with no significant decreases in lysosome movement are the sham and exposure to a single nsPEF in Ca2+-free solution.

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Thermotropic lipid and protein transitions in Chinese hamster lung cell membranes: relationship to hyperthermic cell killing

Canadian Journal of Biochemistry and Cell Biology ◽

10.1139/o83-057 ◽

1983 ◽

Vol 61 (6) ◽

pp. 421-427 ◽

Cited By ~ 65

Author(s):

James R. Lepock ◽

Kwan-Hon Cheng ◽

Hisham Al-Qysi ◽

Jack Kruuv

Keyword(s):

Mammalian Cells ◽

Plasma Membranes ◽

Chinese Hamster ◽

Cell Killing ◽

Water Soluble ◽

Growth Data ◽

Protein Fluorescence ◽

Polar Groups ◽

Spin Resonance ◽

Intrinsic Protein Fluorescence

Exposure of mammalian cells to hyperthermic temperatures (ca. 41–45 °C) appears to act as a direct or triggering effect to produce some later response such as cell death, thermotolerance, or heat-shock protein synthesis. The high activation energy of cell killing indicates that the direct effect of hyperthermia might be a thermotropic transition in some cellular component, for this particular response. Both hyperthermic survival and growth data imply that the temperature for the onset of hyperthermic cell killing is 40–41.5 °C for Chinese hamster lung V79 cells. Studies using the electron spin resonance label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide and the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene show the existence of lipid transitions at approximately 7–8 and 23–36 °C (or a broad transition between these temperatures) in mitochondria and whole cell homogenates, that correlate well with changes in growth and hypothermic killing. No lipid transition was detected near 40–41.5 °C that could correlate with hyperthermic killing in either mitochondrial or plasma membranes, but measurements of intrinsic protein fluorescence and protein fluorophore to trans-paranaric acid energy transfer demonstrate the existence of an irreversible transition in protein structure or arrangement above ca. 40 °C in both mitochondrial and plasma membranes. This transition is due to protein rearrangement and (or) unfolding such that there is increased exposure of protein tryptophan and tyrosine residues to polar groups and to paranaric acid. The strength of the transition implies that a significant fraction of total membrane protein is involved in this transition, which may be analogous to the heat-induced denaturation of water-soluble proteins. This alteration in membrane structure above ca. 40 °C could cause many of the observed changes in plasma membrane and mitochondrial function, which may further be involved in cellular responses to hyperthermia.

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LION/web: a web-based ontology enrichment tool for lipidomic data analysis

GigaScience ◽

10.1093/gigascience/giz061 ◽

2019 ◽

Vol 8 (6) ◽

Cited By ~ 21

Author(s):

Martijn R Molenaar ◽

Aike Jeucken ◽

Tsjerk A Wassenaar ◽

Chris H A van de Lest ◽

Jos F Brouwers ◽

...

Keyword(s):

Membrane Fluidity ◽

Plasma Membranes ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Lateral Diffusion ◽

Decanoic Acid ◽

Web Based ◽

Ovary Cells ◽

Lipid Species ◽

Significant Enrichment

Abstract Background A major challenge for lipidomic analyses is the handling of the large amounts of data and the translation of results to interpret the involvement of lipids in biological systems. Results We built a new lipid ontology (LION) that associates >50,000 lipid species to biophysical, chemical, and cell biological features. By making use of enrichment algorithms, we used LION to develop a web-based interface (LION/web, www.lipidontology.com) that allows identification of lipid-associated terms in lipidomes. LION/web was validated by analyzing a lipidomic dataset derived from well-characterized sub-cellular fractions of RAW 264.7 macrophages. Comparison of isolated plasma membranes with the microsomal fraction showed a significant enrichment of relevant LION-terms including “plasma membrane", “headgroup with negative charge", "glycerophosphoserines", “above average bilayer thickness", and “below average lateral diffusion". A second validation was performed by analyzing the membrane fluidity of Chinese hamster ovary cells incubated with arachidonic acid. An increase in membrane fluidity was observed both experimentally by using pyrene decanoic acid and by using LION/web, showing significant enrichment of terms associated with high membrane fluidity ("above average", "very high", and "high lateral diffusion" and "below average transition temperature"). Conclusions The results demonstrate the functionality of LION/web, which is freely accessible in a platform-independent way.

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

PeerJ ◽

10.7717/peerj.3267 ◽

2017 ◽

Vol 5 ◽

pp. e3267 ◽

Cited By ~ 12

Author(s):

Vitalij Novickij ◽

Janja Dermol ◽

Audrius Grainys ◽

Matej Kranjc ◽

Damijan Miklavčič

Keyword(s):

Mammalian Cells ◽

Fluorescent Dyes ◽

Pulse Generator ◽

Chinese Hamster ◽

Electrical Conductance ◽

Single Pulse ◽

Membrane Permeabilization ◽

Contactless Method ◽

Dependent Manner ◽

Cell Permeabilization

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.

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Mechanistic studies of gene delivery into mammalian cells by electrical short-circuiting via an aqueous droplet in dielectric oil

PLoS ONE ◽

10.1371/journal.pone.0243361 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0243361

Author(s):

Hirofumi Kurita ◽

Hirohito Nihonyanagi ◽

Yuki Watanabe ◽

Kenta Sugano ◽

Ryuto Shinozaki ◽

...

Keyword(s):

Electric Field ◽

Mammalian Cells ◽

Short Circuit ◽

Gene Transfection ◽

Jurkat Cells ◽

Dependent Manner ◽

Exogenous Dna ◽

Membrane Pore ◽

A Cell ◽

Aqueous Droplet

We have developed a novel methodology for the delivery of cell-impermeable molecules, based on electrical short-circuiting via a water droplet in dielectric oil. When a cell suspension droplet is placed between a pair of electrodes with an intense DC electric field, droplet bouncing and droplet deformation, which results in an instantaneous short-circuit, can be induced, depending on the electric field strength. We have demonstrated successful transfection of various mammalian cells using the short-circuiting; however, the molecular mechanism remains to be elucidated. In this study, flow cytometric assays were performed with Jurkat cells. An aqueous droplet containing Jurkat cells and plasmids carrying fluorescent proteins was treated with droplet bouncing or short-circuiting. The short-circuiting resulted in sufficient cell viability and fluorescent protein expression after 24 hours’ incubation. In contrast, droplet bouncing did not result in successful gene transfection. Transient membrane pore formation was investigated by uptake of a cell-impermeable fluorescence dye YO-PRO-1 and the influx of calcium ions. As a result, short-circuiting increased YO-PRO-1 fluorescence intensity and intracellular calcium ion concentration, but droplet bouncing did not. We also investigated the contribution of endocytosis to the transfection. The pre-treatment of cells with endocytosis inhibitors decreased the efficiency of gene transfection in a concentration-dependent manner. Besides, the use of pH-sensitive dye conjugates indicated the formation of an acidic environment in the endosomes after the short-circuiting. Endocytosis is a possible mechanism for the intracellular delivery of exogenous DNA.

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Pulsed Electric Fields Alter Expression of NF-κB Promoter-Controlled Gene

International Journal of Molecular Sciences ◽

10.3390/ijms23010451 ◽

2021 ◽

Vol 23 (1) ◽

pp. 451

Author(s):

Justina Kavaliauskaitė ◽

Auksė Kazlauskaitė ◽

Juozas Rimantas Lazutka ◽

Gatis Mozolevskis ◽

Arūnas Stirkė

Keyword(s):

Gene Expression ◽

Synthetic Biology ◽

Electric Field ◽

Electric Fields ◽

Mammalian Cells ◽

Single Gene ◽

Pulsed Electric Fields ◽

Pulsed Electric Field ◽

Potential Candidate ◽

Cell Parameters

The possibility to artificially adjust and fine-tune gene expression is one of the key milestones in bioengineering, synthetic biology, and advanced medicine. Since the effects of proteins or other transgene products depend on the dosage, controlled gene expression is required for any applications, where even slight fluctuations of the transgene product impact its function or other critical cell parameters. In this context, physical techniques demonstrate optimistic perspectives, and pulsed electric field technology is a potential candidate for a noninvasive, biophysical gene regulator, exploiting an easily adjustable pulse generating device. We exposed mammalian cells, transfected with a NF-κB pathway-controlled transcription system, to a range of microsecond-duration pulsed electric field parameters. To prevent toxicity, we used protocols that would generate relatively mild physical stimulation. The present study, for the first time, proves the principle that microsecond-duration pulsed electric fields can alter single-gene expression in plasmid context in mammalian cells without significant damage to cell integrity or viability. Gene expression might be upregulated or downregulated depending on the cell line and parameters applied. This noninvasive, ligand-, cofactor-, nanoparticle-free approach enables easily controlled direct electrostimulation of the construct carrying the gene of interest; the discovery may contribute towards the path of simplification of the complexity of physical systems in gene regulation and create further synergies between electronics, synthetic biology, and medicine.

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Aster Proteins Regulate the Accessible Cholesterol Pool in the Plasma Membrane

Molecular and Cellular Biology ◽

10.1128/mcb.00255-20 ◽

2020 ◽

Vol 40 (19) ◽

Cited By ~ 2

Author(s):

Alessandra Ferrari ◽

Cuiwen He ◽

John Paul Kennelly ◽

Jaspreet Sandhu ◽

Xu Xiao ◽

...

Keyword(s):

Mammalian Cells ◽

Plasma Membranes ◽

Secondary Ion Mass Spectrometry ◽

Threshold Level ◽

Cell Types ◽

Cholesterol Concentration ◽

Dependent Manner ◽

Transport Pathway ◽

Concentration Dependent Manner ◽

Mouse Macrophages

ABSTRACT Recent studies have demonstrated the existence of a discrete pool of cholesterol in the plasma membranes (PM) of mammalian cells—referred to as the accessible cholesterol pool—that can be detected by the binding of modified versions of bacterial cytolysins (e.g., anthrolysin O). When the amount of accessible cholesterol in the PM exceeds a threshold level, the excess cholesterol moves to the endoplasmic reticulum (ER), where it regulates the SREBP2 pathway and undergoes esterification. We reported previously that the Aster/Gramd1 family of sterol transporters mediates nonvesicular movement of cholesterol from the PM to the ER in multiple mammalian cell types. Here, we investigated the PM pool of accessible cholesterol in cholesterol-loaded fibroblasts with a knockdown of Aster-A and in mouse macrophages from Aster-B and Aster-A/B-deficient mice. Nanoscale secondary ion mass spectrometry (NanoSIMS) analyses revealed expansion of the accessible cholesterol pool in cells lacking Aster expression. The increased accessible cholesterol pool in the PM was accompanied by reduced cholesterol movement to the ER, evidenced by increased expression of SREBP2-regulated genes. Cosedimentation experiments with liposomes revealed that the Aster-B GRAM domain binds to membranes in a cholesterol concentration-dependent manner and that the binding is facilitated by the presence of phosphatidylserine. These studies revealed that the Aster-mediated nonvesicular cholesterol transport pathway controls levels of accessible cholesterol in the PM, as well as the activity of the SREBP pathway.

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Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100095017 ◽

1972 ◽

Vol 30 ◽

pp. 350-351

Author(s):

K. Shankar Narayan ◽

Kailash C. Gupta ◽

Tohru Okigaki

Keyword(s):

Ultraviolet Light ◽

Mammalian Cells ◽

Biological Effects ◽

Survival Rates ◽

Chinese Hamster ◽

Cell Types ◽

Differential Sensitivity ◽

Ultrastructural Changes ◽

Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

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Tuning the Electric Field Response of MOFs by Rotatable Dipolar Linkers

10.26434/chemrxiv.8153198.v1 ◽

2019 ◽

Author(s):

Johannes P. Dürholt ◽

Babak Farhadi Jahromi ◽

Rochus Schmid

Keyword(s):

Electric Field ◽

Electric Fields ◽

Structural Changes ◽

Dielectric Behavior ◽

Two Dimensions ◽

Dielectric Constants ◽

Electric Response ◽

Dipole Strength ◽

Metal Organic ◽

Dynamics Simulations

Recently the possibility of using electric fields as a further stimulus to trigger structural changes in metal-organic frameworks (MOFs) has been investigated. In general, rotatable groups or other types of mechanical motion can be driven by electric fields. In this study we demonstrate how the electric response of MOFs can be tuned by adding rotatable dipolar linkers, generating a material that exhibits paralectric behavior in two dimensions and dielectric behavior in one dimension. The suitability of four different methods to compute the relative permittivity κ by means of molecular dynamics simulations was validated. The dependency of the permittivity on temperature T and dipole strength μ was determined. It was found that the herein investigated systems exhibit a high degree of tunability and substantially larger dielectric constants as expected for MOFs in general. The temperature dependency of κ obeys the Curie-Weiss law. In addition, the influence of dipolar linkers on the electric field induced breathing behavior was investigated. With increasing dipole moment, lower field strength are required to trigger the contraction. These investigations set the stage for an application of such systems as dielectric sensors, order-disorder ferroelectrics or any scenario where movable dipolar fragments respond to external electric fields.

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Meta-Deflectors Made of Dielectric Nanohole Arrays with Anti-Damage Potential

Photonics ◽

10.3390/photonics8040107 ◽

2021 ◽

Vol 8 (4) ◽

pp. 107

Author(s):

Haichao Yu ◽

Feng Tang ◽

Jingjun Wu ◽

Zao Yi ◽

Xin Ye ◽

...

Keyword(s):

Electric Field ◽

Electric Fields ◽

Laser Cutting ◽

High Complexity ◽

Damage Potential ◽

Optical Components ◽

Nanohole Arrays ◽

Intense Light ◽

Polarization Of Light ◽

Air Hole

In intense-light systems, the traditional discrete optical components lead to high complexity and high cost. Metasurfaces, which have received increasing attention due to the ability to locally manipulate the amplitude, phase, and polarization of light, are promising for addressing this issue. In the study, a metasurface-based reflective deflector is investigated which is composed of silicon nanohole arrays that confine the strongest electric field in the air zone. Subsequently, the in-air electric field does not interact with the silicon material directly, attenuating the optothermal effect that causes laser damage. The highest reflectance of nanoholes can be above 99% while the strongest electric fields are tuned into the air zone. One presentative deflector is designed based on these nanoholes with in-air-hole field confinement and anti-damage potential. The 1st order of the meta-deflector has the highest reflectance of 55.74%, and the reflectance sum of all the orders of the meta-deflector is 92.38%. The optothermal simulations show that the meta-deflector can theoretically handle a maximum laser density of 0.24 W/µm2. The study provides an approach to improving the anti-damage property of the reflective phase-control metasurfaces for intense-light systems, which can be exploited in many applications, such as laser scalpels, laser cutting devices, etc.

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Integrating flexible electronics for pulsed electric field delivery in a vascularized 3D glioblastoma model

npj Flexible Electronics ◽

10.1038/s41528-021-00115-x ◽

2021 ◽

Vol 5 (1) ◽

Author(s):

Marie C. Lefevre ◽

Gerwin Dijk ◽

Attila Kaszas ◽

Martin Baca ◽

David Moreau ◽

...

Keyword(s):

Electric Field ◽

Electric Fields ◽

Flexible Electronics ◽

Soft Tissues ◽

Multiphoton Microscopy ◽

Membrane Integrity ◽

Tumor Model ◽

Pulsed Electric Fields ◽

In Ovo ◽

Cell Membrane Integrity

AbstractGlioblastoma is a highly aggressive brain tumor, very invasive and thus difficult to eradicate with standard oncology therapies. Bioelectric treatments based on pulsed electric fields have proven to be a successful method to treat cancerous tissues. However, they rely on stiff electrodes, which cause acute and chronic injuries, especially in soft tissues like the brain. Here we demonstrate the feasibility of delivering pulsed electric fields with flexible electronics using an in ovo vascularized tumor model. We show with fluorescence widefield and multiphoton microscopy that pulsed electric fields induce vasoconstriction of blood vessels and evoke calcium signals in vascularized glioblastoma spheroids stably expressing a genetically encoded fluorescence reporter. Simulations of the electric field delivery are compared with the measured influence of electric field effects on cell membrane integrity in exposed tumor cells. Our results confirm the feasibility of flexible electronics as a means of delivering intense pulsed electric fields to tumors in an intravital 3D vascularized model of human glioblastoma.

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