Flavien Pillet

Cell wall as a target for bacteria inactivation by pulsed electric fields

The integrity and morphology of bacteria is sustained by the cell wall, the target of the main microbial inactivation processes. One promising approach to inactivation is based on the use of pulsed electric fields (PEF). The current dogma is that irreversible cell membrane electro-permeabilisation causes the death of the bacteria. However, the actual effect on the cell-wall architecture has been poorly explored. Here we combine atomic force microscopy and electron microscopy to study the cell-wall organization of living Bacillus pumilus bacteria at the nanoscale. For vegetative bacteria, exposure to PEF led to structural disorganization correlated with morphological and mechanical alterations of the cell wall. For spores, PEF exposure led to the partial destruction of coat protein nanostructures, associated with internal alterations of cortex and core. Our findings reveal for the first time that the cell wall and coat architecture are directly involved in the electro-eradication of bacteria.

Generation of living cell arrays for atomic force microscopy studies.

Atomic force microscopy (AFM) is a useful tool for studying the morphology or the nanomechanical and adhesive properties of live microorganisms under physiological conditions. However, to perform AFM imaging, living cells must be immobilized firmly enough to withstand the lateral forces exerted by the scanning tip, but without denaturing them. This protocol describes how to immobilize living cells, ranging from spores of bacteria to yeast cells, into polydimethylsiloxane (PDMS) stamps, with no chemical or physical denaturation. This protocol generates arrays of living cells, allowing statistically relevant measurements to be obtained from AFM measurements, which can increase the relevance of results. The first step of the protocol is to generate a microstructured silicon master, from which many microstructured PDMS stamps can be replicated. Living cells are finally assembled into the microstructures of these PDMS stamps using a convective and capillary assembly. The complete procedure can be performed in 1 week, although the first step is done only once, and thus repeats can be completed within 1 d.

Dendrimer functionalization of gold surface improves the measurement of protein–DNA interactions by surface plasmon resonance imaging

Surface Plasmon Resonance imaging (SPRi) is a label free technique typically used to follow biomolecular interactions in real time. SPRi offers the possibility to simultaneously investigate numerous interactions and is dedicated to high throughput analysis. However, precise determination of binding constants between partners is not highly reliable. We report here a dendrimer functionalization of gold surface that significantly improves selectivity of the detection of protein-DNA interactions. We showed that amino-gold surface functionalization with phosphorus dendrimers of fourth generation (G4) allowed complete coverage of the gold surface and the increase of the surface roughness. We optimized the conditions for DNA probe deposition to allow accurate detection of a well-known protein-DNA interaction involved in bacterial chromosome segregation. Using this G4-functionalized surface, the specificity of the SPRi response was significantly improved allowing discrimination between protein and DNA interactions of different strengths. Kinetic constants similar to those obtained with other techniques currently used in molecular biology were only obtained with the G4 dendrimer functionalized surface. This study demonstrated the benefit of using dendrimeric surfaces for sensitive high throughput SPRi analysis.

Shock waves associated with electric pulses affect cell electro-permeabilization.

New features of cell electro-permeabilization are obtained by using high field (several tens of kV/cm) with short (sub-microsecond, nanosecond) pulse duration. Arcing appears as a main safety problem when air gaps are present between electrodes. A new applicator design was chosen to obtain a closed chamber where high field pulses could be delivered in a safe way with very short pulse duration. The safety issue of the system was validated under millisecond, microsecond and nanosecond pulses. The closed chamber applicator was then checked for its use under classical electro-mediated permeabilization and electro-gene transfer (EGT). A 20 times decrease in gene expression was observed compared with classical open chambers. It was experimentally observed that shock waves were present under the closed chamber configuration of the applicator. This was not the case with an open chamber design. Electropulsation chamber design plays a role on pulsing conditions and in the efficiency of gene electro transfer.

Generator and Setup for Emulating Exposures of Biological Samples to Lightning Strokes

Goal: We aimed to develop a system for controlled exposure of biological samples to conditions they experience when lightning strikes their habitats. Methods: We based the generator on a capacitor charged via a bridge rectifier and a dc-dc converter, and discharged via a relay, delivering arcs similar to natural lightning strokes in electric current waveform and similarly accompanied by acoustic shock waves. We coupled the generator to our exposure chamber described previously, measured electrical and acoustic properties of arc discharges delivered, and assessed their ability to inactivate bacterial spores. Results: Submicrosecond discharges descended vertically from the conical emitting electrode across the air gap, entering the sample centrally and dissipating radially toward the ring-shaped receiving electrode. In contrast, longer discharges tended to short-circuit the electrodes. Recording at 341 000 FPS with Vision Research Phantom v2010 camera revealed that initial arc descent was still vertical, but became accompanied by arcs leaning increasingly sideways; after 8-12 μs, as the first of these arcs formed direct contact with the receiving electrode, it evolved into a channel of plasmified air and short-circuited the electrodes. We eliminated this artefact by incorporating an insulating cylinder concentrically between the electrodes, precluding short-circuiting between them. While bacterial spores are highly resistant to electric pulses delivered through direct contact, we showed that with arc discharges accompanied by an acoustic shock wave, spore inactivation is readily obtained. Conclusion: The presented system allows scientific investigation of effects of arc discharges on biological samples. Significance: This system will allow realistic experimental studies of lightning-triggered horizontal gene transfer and assessment of its role in evolution.

Importance of endogenous extracellular matrix in biomechanical properties of human skin model

The physical and mechanical properties of cells modulate their behavior such proliferation rate, migration and extracellular matrix remodeling. In order to study cell behavior in a tissue-like environment in vitro, it is of utmost importance to develop biologically and physically relevant 3D cell models. Here, we characterized the physical properties of a single cell type growing in configurations of increasing complexity. From one human skin biopsy, primary dermal fibroblasts were isolated and seeded to give monolayer (2D model), spheroid (3D model poor in extracellular matrix) and tissue-engineered cell sheet (3D model rich in endogenous extracellular matrix). Living native human dermis tissue was used as a gold standard. Nanomechanical and viscoelastic properties at the cell scale were measured by atomic force microscopy (AFM) while biphoton microscopy allowed collagen detection by second harmonic generation and scanning electron microscopy helped in model morphological characterization. In all models, fibroblasts presented a similar typical elongated cell shape, with a cytoskeleton well-arranged along the long axis of the cell. However, elastic moduli of the tissue-engineered cell sheet and native dermis tissue were similar and statistically lower than monolayer and spheroid models. We successfully carried out AFM force measurements on 3D models such as spheroids and tissue-engineered cell sheets, as well as on living native human tissue. We demonstrated that a tissue-engineered dermal model recapitulates the mechanical properties of human native dermal tissue unlike the classically used monolayer and spheroid models. Furthermore, we give statistical evidence to indicate a correlation between cell mechanical properties and the presence of collagens in the models studied.

Inactivation of spores by electric arcs

BackgroundIn the context of spore contamination involved in bio-terrorism and food preservation, the development of new techniques for spore inactivation is an important challenge.ResultsHere, a successful application of electric arc discharges resulting in spore death was reported. Two types of electric arcs were compared, different with respect to their durations. The discharges with 0.5 μs duration induced a small inactivation area of 0.6 % of surface treated around their point of entry into the sample, while those with 20 μs duration induced a much larger inactivation area from 7 to 55 % of surface treated roughly proportional to the number of discharges delivered. In particular, 50 discharges of 20 μs duration induced inactivation in more than 55% of surface treated at an inactivation rate above 3.6 log10.ConclusionsThese results are promising and warrant developing electric arcing as a novel method for spore inactivation.

Plane wave in vitro exposure of biological samples, geometries considerations

This paper deals with in vitro experiments involving biological samples exposed to free space high power pulsed electromagnetic plane waves. Biological samples under test are Giant Unilamellar Vesicles (GUV) in solutions or human mammalian tumor cells in suspensions. Different sample containers (such as Petri dishes, Eppendorf tubes or microscope glass slides) have been evaluated in terms of reachable electric coupling efficiency and reachable electric field homogeneity in the biological samples under study. Recommendations for the use of each of them are suggested. Due to the high permittivity of the cell culture medium, we show that the Device Under Test (DUT) may behave as a dielectric resonator. It is the case for the Petri Dish configuration with H field coupling in the axis direction for which the TE01δ dielectric mode can be excited. At last we present a new idea for Field Homogenization by Volume Extension (FHVE).

High power electromagnetic pulse applicators for evaluation of biological effects induced by electromagnetic radiation waves

The effects of electromagnetic radiation waves on health is one of the major public concerns. These waves are mainly produced at a large scale but it is important to evaluate these effects on biological samples at the laboratory scale. Here we developed a set of micro applicators, which allow evaluating the effect of electromagnetic fields on biological samples with volumes in the microliter range. The applicators can be coupled to an optical microscope and allow a real-time observation of potential structural and functional alterations of the tested sample induced by different waveforms. New design approaches are suggested to simultaneously achieve maximized electric field coupling effect and optimized electric field homogeneity in the tested sample, while minimizing the return loss when the applicators are loaded with the biological samples. These applicators allow studying the biological effect of a variety of different signals, due to their wide frequency bandwidth (beyond 1.5 GHz) and their high permissible power. In addition, different electromagnetic parameters such as the electromagnetic field magnitude, pulse repetitive factor, number of bursts or delay between bursts may be set. The efficacy of the applicators was addressed for three different signals: two types of electromagnetic waves – a damped sinusoid centered at 200 MHz (wide band signal), a radar-like signal at 1.5 GHz (the ultra-narrow band signal) and a train of millisecond square-wave monopolar electric field pulses (causing electroporation). The biological effects were thus assessed (at the microscopic scale) on two different biological models, the giant unilamellar vesicles, and tumor and normal human cells, as well as being compared to results obtained (at full scale) with signals generated by antennas.

Electrical discharges in water induce spores’ DNA damage

Bacterial spores are one of the most resilient life forms on earth and are involved in many human diseases, such as infectious diarrhea, fatal paralytic illnesses and respiratory infections. Here, we investigated the mechanisms involved in the death of Bacillus pumilus spores after exposure to electric arcs in water. Cutting-edge microscopies at the nanoscale did not reveal any structural disorganization of spores exposed to electric arcs. This result suggested the absence of physical destruction by a propagating shock wave or an exposure to an electric field. However, Pulsed-Field Gel Electrophoresis (PFGE) revealed genomic DNA damage induced by UV radiation and Reactive Oxygen Species (ROS). UV induced single-strand DNA breaks and thymine dimers while ROS were mainly involved in base excision. Our findings revealed a correlation between DNA damage and the treatment of spores with electrical discharges.