Saint-Léonard-de-Noblat, France
Saint-Léonard-de-Noblat, France

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Coustets M.,CNRS Institute of Pharmacology and Structural Biology | Coustets M.,Toulouse 1 University Capitole | Joubert-Durigneux V.,CERPEM | Herault J.,University of Maine, France | And 5 more authors.
Bioelectrochemistry | Year: 2015

Classical methods, used for large scale treatments such as mechanical or chemical extractions, affect the integrity of extracted cytosolic protein by releasing proteases contained in vacuoles. Our previous experiments on flow processes electroextraction on yeasts proved that pulsed electric field technology allows preserving the integrity of released cytosolic proteins, by not affecting vacuole membranes. Furthermore, large cell culture volumes are easily treated by the flow technology. Based on this previous knowledge, we developed a new protocol in order to electro-extract total cytoplasmic proteins from microalgae (Nannochloropsis salina, Chlorella vulgaris and Haematococcus pluvialis). Given that induction of electropermeabilization is under the control of target cell size, as the mean diameter for N. salina is only 2.5. μm, we used repetitive 2. ms long pulses of alternating polarities with stronger field strengths than previously described for yeasts. The electric treatment was followed by a 24. h incubation period in a salty buffer. The amount of total protein release was observed by a classical Bradford assay. A more accurate evaluation of protein release was obtained by SDS-PAGE. Similar results were obtained with C. vulgaris and H. pluvialis under milder electrical conditions as expected from their larger size. © 2014 Elsevier B.V.


Guionet A.,CNRS Institute of Pharmacology and Structural Biology | Guionet A.,Toulouse 1 University Capitole | David F.,VERI | Zaepffel C.,ONERA | And 8 more authors.
Bioelectrochemistry | Year: 2014

One of the different ways to eradicate microorganisms, and particularly bacteria that might have an impact on health consists in the delivery of pulsed electric fields (PEFs). The technologies of millisecond (ms) or microsecond (μs) PEF are still well known and used for instance in the process of fruit juice sterilization. However, this concept is costly in terms of delivered energy which might be too expensive for some other industrial processes.Nanosecond pulsed electric fields (nsPEFs) might be an alternative at least for lower energetic cost. However, only few insights were available and stipulate a gain in cost and in efficiency as well. Using Escherichia coli, the impact of frequency and low rate on eradication and energy consumption by msPEF, μsPEF and nsPEF have been studied and compared. While a 1 log10 was reached with an energy cost of 100 and 158kJ/L with micro- and millisecond PEFs respectively, nsPEF reached the reduction for similar energy consumption. The best condition was obtained for a 1 log10 deactivation in 0.5h, for energy consumption of 143kJ/L corresponding to 0.04W·h when the field was around 100kV/cm. Improvement can also be expected by producing a generator capable to increase the electric field. © 2014 Elsevier B.V.


Coustets M.,CNRS Institute of Pharmacology and Structural Biology | Coustets M.,Toulouse 1 University Capitole | Joubert-Durigneux V.,CERPEM | He rault J.,University of Maine, France | And 5 more authors.
Bioelectrochemistry | Year: 2014

Classical methods, used for large scale treatments such as mechanical or chemical extractions, affect the integrity of extracted cytosolic protein by releasing proteases contained in vacuoles. Our previous experiments on flow processes electroextraction on yeasts proved that pulsed electric field technology allows preserving the integrity of released cytosolic proteins, by not affecting vacuole membranes. Furthermore, large cell culture volumes are easily treated by the flow technology. Based on this previous knowledge, we developed a new protocol in order to electro-extract total cytoplasmic proteins from microalgae (Nannochloropsis salina, Chlorella vulgaris and Haematococcus pluvialis). Given that induction of electropermeabilization is under the control of target cell size, as the mean diameter for N. salina is only 2.5 μm, we used repetitive 2 ms long pulses of alternating polarities with stronger field strengths than previously described for yeasts. The electric treatment was followed by a 24. h incubation period in a salty buffer. The amount of total protein release was observed by a classical Bradford assay. A more accurate evaluation of protein release was obtained by SDS-PAGE. Similar results were obtained with C. vulgaris and H. pluvialis under milder electrical conditions as expected from their larger size. © 2014 Elsevier B.V.


PubMed | University of Maine, France, CERPEM and Hunter Medical Research Institute
Type: Journal Article | Journal: International journal of oncology | Year: 2015

Docosahexaenoic acid (DHA), the main member of the omega-3 essential fatty acid family, has been shown to reduce the invasion of the triple-negative breast cancer cell line MDA-MB-231, but the mechanism involved remains unclear. In the present study, a proteomic approach was used to define changes in protein expression induced by DHA. Proteins from crude membrane preparations of MDA-MB-231 cells treated with 100 M DHA were separated by two-dimensional electrophoresis (2-DE) and differentially expressed proteins were identified using MALDI-TOF mass spectrometry. The main changes observed were the upregulation of Keratin, type cytoskeletal 1 (KRT1), catalase and lamin-A/C. Immunocytochemistry analyses confirmed the increase in KRT1 induced by DHA. Furthermore, in vitro invasion assays showed that siRNA against KRT1 was able to reverse the DHA-induced inhibition of breast cancer cell invasion. In conclusion, KRT1 is involved in the anti-invasive activity of DHA in breast cancer cells.


Guionet A.,CNRS Institute of Pharmacology and Structural Biology | Guionet A.,Toulouse 1 University Capitole | David F.,VERI | Zaepffel C.,ONERA | And 8 more authors.
Bioelectrochemistry | Year: 2015

One of the different ways to eradicate microorganisms, and particularly bacteria that might have an impact on health consists in the delivery of pulsed electric fields (PEFs). The technologies of millisecond (ms) or microsecond (μs) PEF are still well known and used for instance in the process of fruit juice sterilization. However, this concept is costly in terms of delivered energy which might be too expensive for some other industrial processes.Nanosecond pulsed electric fields (nsPEFs) might be an alternative at least for lower energetic cost. However, only few insights were available and stipulate a gain in cost and in efficiency as well. Using Escherichia coli, the impact of frequency and low rate on eradication and energy consumption by msPEF, μsPEF and nsPEF have been studied and compared. While a 1 log10 was reached with an energy cost of 100 and 158kJ/L with micro- and millisecond PEFs respectively, nsPEF reached the reduction for similar energy consumption. The best condition was obtained for a 1 log10 deactivation in 0.5h, for energy consumption of 143kJ/L corresponding to 0.04W·h when the field was around 100kV/cm. Improvement can also be expected by producing a generator capable to increase the electric field. © 2014 Elsevier B.V.


Joubert V.,CERPEM | Cheype C.,CERPEM | Bonnet J.,ONERA | Packan D.,ONERA | And 4 more authors.
Water Research | Year: 2013

Spores are dormant units of bacteria resistant to numerous disinfection methods. Additionally, the effects on bacteria of repetitive electrical discharges in water by used of the so-called " corona discharges" or streamer are poorly described. In this study vegetative and spore forms of Bacillus subtilis var. niger were subjected to these discharges. To generate corona discharges in water, a Marx generator capable of delivering 60-90 kV was used with a coaxial chamber of treatment. Vegetative and spore form reductions were defined using colony-forming unit counting. Proteins extracts were separated by two-dimensional electrophoresis and spots of interest were characterized by mass spectrometry. Shock waves were assessed by the diminution of liposome size and OD400 nm. The results show a decrease in bacteria viability of 2 log10 after 1000 discharges on the vegetative form and 4 log10 after 10,000 discharges on the spores. Two-dimensional electrophoresis showed that the streamers impact the regulation of several proteins in the vegetative forms with UniProt ID: P80861, Q06797, P80244, C0ZI91, respectively. The reduction appears to be due, in part, to hydrogen peroxide (H2O2) generated by the corona discharges while spore deactivation remained insensitive to these chemicals. The spore eradication was associated to shock waves induced by the discharges but not H2O2. Corona discharges appear as a prospective method for eradication of spores in water. The corona discharges can be an efficient method for decontamination processes of waste water. © 2012 Elsevier Ltd.


Blanckaert V.,CNRS Sea, Molecules and Health | Blanckaert V.,British Petroleum | Kerviel V.,CNRS Sea, Molecules and Health | Kerviel V.,British Petroleum | And 5 more authors.
International Journal of Oncology | Year: 2015

Docosahexaenoic acid (DHA), the main member of the omega-3 essential fatty acid family, has been shown to reduce the invasion of the triple-negative breast cancer cell line MDA-MB-231, but the mechanism involved remains unclear. In the present study, a proteomic approach was used to define changes in protein expression induced by DHA. Proteins from crude membrane preparations of MDA-MB-231 cells treated with 100 μM DHA were separated by two-dimensional electrophoresis (2-DE) and differentially expressed proteins were identified using MALDI-TOF mass spectrometry. The main changes observed were the upregulation of Keratin, type II cytoskeletal 1 (KRT1), catalase and lamin-A/C. Immunocytochemistry analyses confirmed the increase in KRT1 induced by DHA. Furthermore, in vitro invasion assays showed that siRNA against KRT1 was able to reverse the DHA-induced inhibition of breast cancer cell invasion. In conclusion, KRT1 is involved in the anti-invasive activity of DHA in breast cancer cells. © 2015, Spandidos Publications. All rights reserved.

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