Metal-coated plastics recycling by pulsed electric discharge.

Recycling of useful materials such as metals and plastics are evaluated important from the viewpoint of resource conservation and environmental protection. In this respect, the application of pulsed power technology to the recycling field has attracted considerable attention. We used a compact disc-read only memory (CD-ROM) as a separation processing target in this study. A magnetic pulse compression pulsed power generator (MPC-PPG) was designed and constructed to provide a positive pulse voltage. By applying an electric discharge, the protective layer containing the metal layer was separated from the plastic substrate in the atmospheric air. Here, to clarify the mechanism of the metal separation, shock waves and their induced fluid flow generation and propagation were observed by schlieren and shadowgraph visualization methods. Initially, the Mach number of the shock wave was 5.6, soon afterward the shock wave velocity decreased gradually. The fragments of the metal and the protective layers were removed from the plastic substrate with the shock wave propagation. The proposed method and process is applicable to the recycling of electronic wastes on an industrial scale for efficient recovery of valuable materials.

Medium's conductivity and stage of growth as crucial parameters for efficient hydrocarbon extraction by electric field from colonial micro-algae.

The green algae Botryococcus braunii produces a high amount of extracellular hydrocarbon, making it a promising algae in the field of bio-fuels production. As it mainly produces squalene like hydrocarbons, cosmetic industries are also interested in its milking. Pulsed electric fields (PEF) are an innovative method allowing oil extraction from micro-algae. In common algae accumulating hydrocarbon inside cytoplasm (Chlorella vulgaris, Nannochloropsis sp., etc), electric fields can destroy cell membranes, allowing the release of hydrocarbon. However, for B.braunii, hydrocarbons adhere to the cell wall outside of cells as a matrix. In a previous article we reported that electric fields can unstick cells from a matrix, allowing hydrocarbon harvesting. In this work, we deeper investigated this phenomenon of cell hatching by following 2 parameters: the conductivity of the medium and the cultivation duration of the culture. Cell hatching is accurately evaluated by both microscopic and macroscopic observations. For high conductivity and a short time of cultivation, almost no effect is observed even after up to 1000 PEF pulses are submitted to the cells. While lower conductivity and a longer cultivation period allow strong cell hatching after 200 PEF pulses are applied to the cells. We identify 2 new crucial parameters, able to turn the method from inefficient to very efficient. It might help companies to save energy and money in case of mass production.

Spatio-temporal dynamics of calcium electrotransfer during cell membrane permeabilization.

Pulsed electric fields (PEFs) are applied as physical stimuli for DNA/drug delivery, cancer therapy, gene transformation, and microorganism eradication. Meanwhile, calcium electrotransfer offers an interesting approach to treat cancer, as it induces cell death easier in malignant cells than in normal cells. Here, we study the spatial and temporal cellular responses to 10 µs duration PEFs; by observing real-time, the uptake of extracellular calcium through the cell membrane. The experimental setup consisted of an inverted fluorescence microscope equipped with a color high-speed framing camera and a specifically designed miniaturized pulsed power system. The setup allowed us to accurately observe the permeabilization of HeLa S3 cells during application of various levels of PEFs ranging from 0.27 to 1.80 kV/cm. The low electric field experiments confirmed the threshold value of transmembrane potential (TMP). The high electric field observations enabled us to retrieve the entire spatial variation of the permeabilization angle (θ). The temporal observations proved that after a minimal permeabilization of the cell membrane, the ionic diffusion was the prevailing mechanism of the delivery to the cell cytoplasm. The observations suggest 0.45 kV/cm and 100 pulses at 1 kHz as an optimal condition to achieve full calcium concentration in the cell cytoplasm. The results offer precise levels of electric fields to control release of the extracellular calcium to the cell cytoplasm for inducing minimally invasive cancer calcium electroporation, an interesting affordable method to treat cancer patients with minimum side effects.

A new mechanism for efficient hydrocarbon electro-extraction from Botryococcus braunii.

BACKGROUND: Recent understanding that specific algae have high hydrocarbon production potential has attracted considerable attention. Botryococcus braunii is a microalga with an extracellular hydrocarbon matrix, which makes it an appropriate green energy source. RESULTS: This study focuses on extracting oil from the microalgae matrix rather than the cells, eliminating the need for an excessive electric field to create electro-permeabilization. In such a way, technical limitations due to high extraction energy and cost can be overcome. Here, nanosecond pulsed electric fields (nsPEF) with 80 ns duration and 20-65 kV/cm electric fields were applied. To understand the extraction mechanism, the structure of the algae was accurately studied under fluorescence microscope; extraction was quantified using image analysis; quality of extraction was examined by thin-layer chromatography (TLC); and the cell/matrix separation was observed real-time under a microscope during nsPEF application. Furthermore, optimization was carried out by screening values of electric fields, pulse repetition frequencies, and energy spent. CONCLUSIONS: The results offer a novel method applicable for fast and continues hydrocarbon extraction process at low energy cost.

Reparable Cell Sonoporation in Suspension: Theranostic Potential of Microbubble.

The conjunction of low intensity ultrasound and encapsulated microbubbles can alter the permeability of cell membrane, offering a promising theranostic technique for non-invasive gene/drug delivery. Despite its great potential, the biophysical mechanisms of the delivery at the cellular level remains poorly understood. Here, the first direct high-speed micro-photographic images of human lymphoma cell and microbubble interaction dynamics are provided in a completely free suspension environment without any boundary parameter defect. Our real-time images and theoretical analyses prove that the negative divergence side of the microbubble's dipole microstreaming locally pulls the cell membrane, causing transient local protrusion of 2.5 µm in the cell membrane. The linear oscillation of microbubble caused microstreaming well below the inertial cavitation threshold, and imposed 35.3 Pa shear stress on the membrane, promoting an area strain of 0.12%, less than the membrane critical areal strain to cause cell rupture. Positive transfected cells with pEGFP-N1 confirm that the interaction causes membrane poration without cell disruption. The results show that the overstretched cell membrane causes reparable submicron pore formation, providing primary evidence of low amplitude (0.12 MPa at 0.834 MHz) ultrasound sonoporation mechanism.

In vivo effects of focused shock waves on tumor tissue visualized by fluorescence staining techniques.

Shock waves can cause significant cytotoxic effects in tumor cells and tissues both in vitro and in vivo. However, understanding the mechanisms of shock wave interaction with tissues is limited. We have studied in vivo effects of focused shock waves induced in the syngeneic sarcoma tumor model using the TUNEL assay, immunohistochemical detection of caspase-3 and hematoxylin-eosin staining. Shock waves were produced by a multichannel pulsed-electrohydraulic discharge generator with a cylindrical ceramic-coated electrode. In tumors treated with shock waves, a large area of damaged tissue was detected which was clearly differentiated from intact tissue. Localization and a cone-shaped region of tissue damage visualized by TUNEL reaction apparently correlated with the conical shape and direction of shock wave propagation determined by high-speed shadowgraphy. A strong TUNEL reaction of nuclei and nucleus fragments in tissue exposed to shock waves suggested apoptosis in this destroyed tumor area. However, specificity of the TUNEL technique to apoptotic cells is ambiguous and other apoptotic markers (caspase-3) that we used in our study did not confirmed this observation. Thus, the generated fragments of nuclei gave rise to a false TUNEL reaction not associated with apoptosis. Mechanical stress from high overpressure shock wave was likely the dominant pathway of tumor damage.

Different involvement of extracellular calcium in two modes of cell death induced by nanosecond pulsed electric fields.

Exposure of cultured cells to nanosecond pulsed electric fields (nsPEFs) induces various cellular responses, including the influx of extracellular Ca2+ and cell death. Recently, nsPEFs have been regarded as a novel means of cancer therapy, but their molecular mechanism of action remains to be fully elucidated. Here, we demonstrate the involvement of extracellular Ca2+ in nsPEF-induced cell death. Extracellular Ca2+ was essential for necrosis and consequent poly(ADP-ribose) (PAR) formation in HeLa S3 cells. Treatment with a Ca2+ ionophore enhanced necrosis as well as PAR formation in nsPEF-exposed HeLa S3 cells. In the absence of extracellular Ca2+, HeLa S3 cells were less susceptible to nsPEFs and exhibited apoptotic proteolysis of caspase 3 and PARP-1. HeLa S3 cells retained the ability to undergo apoptosis even after nsPEF exposure but instead underwent necrosis, suggesting that necrosis is the preferential mode of cell death. In K562 and HEK293 cells, exposure to nsPEFs resulted in the formation of necrosis-associated PAR, whereas Jurkat cells exclusively underwent apoptosis independently of extracellular Ca2+. These observations demonstrate that the mode of cell death induced by nsPEFs is cell-type dependent and that extracellular Ca2+ is a critical factor for nsPEF-induced necrosis.

Nanosecond pulsed electric field suppresses development of eyes and germ cells through blocking synthesis of retinoic acid in Medaka (Oryzias latipes).

Application of nanosecond pulsed electric fields (nsPEFs) has attracted rising attention in various scientific fields including medical, pharmacological, and biological sciences, although its effects and molecular mechanisms leading to the effects remain poorly understood. Here, we show that a single, high-intensity (10-30 kV/cm), 60-ns PEF exposure affects gene expression and impairs development of eyes and germ cells in medaka (Oryzias latipes). Exposure of early blastula stage embryos to nsPEF down-regulated the expression of several transcription factors which are essential for eye development, causing abnormal eye formation. Moreover, the majority of the exposed genetic female embryos showed a fewer number of germ cells similar to that of the control (unexposed) genetic male at 9 days post-fertilization (dpf). However, all-trans retinoic acid (atRA) treatment following the exposure rescued proliferation of germ cells and resumption of normal eye development, suggesting that the phenotypes induced by nsPEF are caused by a decrease of retinoic acid levels. These results confirm that nsPEFs induce novel effects during embryogenesis in medaka.

Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells.

Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs.

Nanosecond pulsed electric fields activate AMP-activated protein kinase: implications for calcium-mediated activation of cellular signaling.

Nanosecond pulsed electric fields (nsPEFs) are increasingly being recognized as a potential tool for use in the life sciences. Exposure of human cells to nsPEFs elicits the formation of small membrane pores, intracellular Ca(2+) mobilization, signaling pathway activation, and apoptosis. Here we report the activation of AMP-activated protein kinase (AMPK) by nsPEFs. AMPK activation is generally achieved by the phosphorylation of AMPK in response to changes in cellular energy status and is mediated by two protein kinases, LKB1 and CaMKK. Exposure to nsPEFs rapidly induced phosphorylation of AMPK and its downstream target ACC in both LKB1-proficient and LKB1-deficient cells. In LKB1-deficient cells, AMPK activation by nsPEFs was mediated by CaMKK and required extracellular Ca(2+), which suggested the occurrence of Ca(2+) mobilization and its participation in AMPK activation by nsPEFs. Our results provide experimental evidence for a direct link between activated cellular signaling and Ca(2+) mobilization in nsPEF-exposed cells.

Nanosecond pulsed electric fields act as a novel cellular stress that induces translational suppression accompanied by eIF2α phosphorylation and 4E-BP1 dephosphorylation.

Recent advances in electrical engineering enable the generation of ultrashort electric fields, namely nanosecond pulsed electric fields (nsPEFs). Contrary to conventional electric fields used for DNA electroporation, nsPEFs can directly reach intracellular components without membrane destruction. Although nsPEFs are now recognized as a unique tool in life sciences, the molecular mechanism of nsPEF action remains largely unclear. Here, we present evidence that nsPEFs act as a novel cellular stress. Exposure of HeLa S3 cells to nsPEFs quickly induced phosphorylation of eIF2α, activation of its upstream stress-responsive kinases, PERK and GCN2, and translational suppression. Experiments using PERK- and GCN2-knockout cells demonstrated dual contribution of PERK and GCN2 to nsPEF-induced eIF2α phosphorylation. Moreover, nsPEF exposure yielded the elevated GADD34 expression, which is known to downregulate the phosphorylated eIF2α. In addition, nsPEF exposure caused a rapid decrease in 4E-BP1 phosphorylation irrespective of the PERK/GCN2 status, suggesting participation of both eIF2α and 4E-BP1 in nsPEF-induced translational suppression. RT-PCR analysis of stress-inducible genes demonstrated that cellular responses to nsPEFs are distinct from those induced by previously known forms of cellular stress. These results provide new mechanistic insights into nsPEF action and implicate the therapeutic potential of nsPEFs for stress response-associated diseases.

Nanosecond pulsed electric fields activate MAPK pathways in human cells.

Application of nanosecond pulsed electric fields (nsPEFs) has attracted attention as a unique tool in life sciences, especially for cancer therapy, but the molecular mechanism of its action on living organisms is yet to be fully elucidated. Here, we report a transient activation of signaling pathways involving mitogen-activated protein kinases (MAPKs) by nsPEFs. Application of nsPEFs to HeLa S3 cells induced phosphorylation of MAPKs, including p38, JNK and ERK, and their upstream kinases. The application of nsPEFs also elicited elevated phosphorylation of downstream factors including MSK1, Hsp27, ATF2, p90RSK, and c-Jun. In addition, the application of nsPEFs led to the transcriptional activation of immediate early genes in the MAPK pathways. Treatment with inhibitors of the MAPK pathways suppressed nsPEF-induced protein phosphorylation and gene expression downstream of MAPKs, confirming the functional connection between the nsPEF-activated MAPKs and the observed induction of the downstream events. Taken together, these results provide important clues to the action of nsPEFs on human cells and demonstrate a new possibility for the utilization of nsPEFs in the control of various biological phenomena involving activation of the MAPK pathways.

Optical observation of cell sonoporation with low intensity ultrasound.

Sonoporation is a promising drug delivery technique with great potential in medicine. However, its applications have been limited mostly by the lack of understanding its underlying biophysical mechanism, partly due to the inadequacy of the existing models for coupling with highly sensitive imaging techniques to directly observe the actual precursor events of cell-microbubble interaction under low intensity ultrasound. Here, we introduce a new in vitro method utilizing capillary-microgripping system and micro-transducer to achieve maximum level of experimental flexibility for capturing real time highly magnified images of cell-microbubble interaction, hitherto unseen in this context. Insonation of isolated single cells and microbubbles parallel with high speed microphotography and fluorescence microscopy allowed us to identify dynamic responses of cell-membrane/microbubble in correlation with sonoporation. Our results showed that bubble motion and linear oscillation in close contact with the cell membrane can cause local deformation and transient porosity in the cell membrane without rupturing it. This method can also be used as an in situ gene/drug delivery system of targeted cells for non-invasive clinical applications.

Activation of the JNK pathway by nanosecond pulsed electric fields.

Nanosecond pulsed electric fields (nsPEFs) are increasingly recognized as a novel and unique tool in various life science fields, including electroporation and cancer therapy, although their mode of action in cells remains largely unclear. Here, we show that nsPEFs induce strong and transient activation of a signaling pathway involving c-Jun N-terminal kinase (JNK). Application of nsPEFs to HeLa S3 cells rapidly induced phosphorylation of JNK1 and MKK4, which is located immediately upstream of JNK in this signaling pathway. nsPEF application also elicited increased phosphorylation of c-Jun protein and dramatically elevated c-jun and c-fos mRNA levels. nsPEF-inducible events downstream of JNK were markedly suppressed by the JNK inhibitor SP600125, which confirmed JNK-dependency of these events in this pathway. Our results provide novel mechanistic insights into the mode of nsPEF action in human cells.

A novel material incorporation technique for medaka (Oryzias latipes) eggs using nanosecond pulsed electric fields.

The effects of nanosecond pulsed electric fields (nsPEFs) on medaka eggs were examined. Although embryogenesis was not affected by nsPEF treatment alone, significant harmful effects were observed when the eggs were treated with nsPEFs in the presence of cycloheximide in the outer solution. Nanosecond PEF treatment affected the permeability of both the egg envelope (chorion) and the cell membrane, which resulted in intracellular incorporation of cycloheximide.

Molecular mechanism of protein assembly on DNA double-strand breaks in the non-homologous end-joining pathway.

Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) in mammalian species. Upon DSB induction, a living cell quickly activates the NHEJ pathway comprising of multiple molecular events. However, it has been difficult to analyze the initial phase of DSB responses in living cells, primarily due to technical limitations. Recent advances in real-time imaging and site-directed DSB induction using laser microbeam allow us to monitor the spatiotemporal dynamics of NHEJ factors in the immediate-early phase after DSB induction. These new approaches, together with the use of cell lines deficient in each essential NHEJ factor, provide novel mechanistic insights into DSB recognition and protein assembly on DSBs in the NHEJ pathway. In this review, we provide an overview of recent progresses in the imaging analyses of the NHEJ core factors. These studies strongly suggest that the NHEJ core factors are pre-assembled into a large complex on DSBs prior to the progression of the biochemical reactions in the NHEJ pathway. Instead of the traditional step-by-step assembly model from the static view of NHEJ, a novel model for dynamic protein assembly in the NHEJ pathway is proposed. This new model provides important mechanistic insights into the protein assembly at DSBs and the regulation of DSB repair.

Cernunnos/XLF: a new player in DNA double-strand break repair.

Non-homologous end-joining (NHEJ) is the predominant repair pathway for DNA double-strand breaks (DSBs) in vertebrates and also plays a crucial role in V(D)J recombination of immunoglobulin genes. Cernunnos/XLF is a newly identified core factor for NHEJ, and its defect causes a genetic disease characterized by neural disorders, immunodeficiency and increased radiosensitivity. Cernunnos/XLF has at least two distinct functions in NHEJ. Cernunnos/XLF interacts with and stimulates the XRCC4/DNA ligase IV complex, which acts at the final ligation step in NHEJ. In living cells, Cernunnos/XLF quickly responds to DSB induction and accumulates at damaged sites in a Ku-dependent but XRCC4-independent manner. These observations indicate that Cernunnos/XLF plays a unique role in bridging damage sensing and DSB rejoining steps of NHEJ. Recent crystallographic analyses of the homodimeric Cernunnos/XLF protein provide structural insights into the Cernunnos/XLF functions. These studies offer important clues toward understanding the molecular mechanism for NHEJ-defective diseases.

A method of water-bloom prevention using underwater pulsed streamer discharge.

Water-bloom (also named as cyanobacterial bloom) is becoming a very serious pollution problem all over the world. In this paper, a new method for the prevention of water blooms using underwater streamer discharges is reported. Blumlein pulse forming network (B-PFN) and magnetic pulse compression circuit (MPC) were employed to apply high voltage pulses to water with cyanobacterial cells. The experimental results confirmed that the cyanobacterial cells sank to the bottom of the water bodies after applying underwater streamer discharges. Transmission electron microscope (TEM) observations showed that the discharge collapsed the gas vesicles (GVs)-the intercellular structure of water-bloom forming cyanobaterial cells-and did not affect the other part of contents of the cells. Cynabacterial cells lost buoyancy and sank to the bottom of the water bodies. Because of lower temperature and without enough sunlight at the bottom of the water bodies, the cells can be prevented from proliferation too quickly.