Jean-Marie Perrier-Cornet

Visualization of RNA-Quadruplexes in Live Cells

Visualization of DNA and RNA quadruplex formation in human cells was demonstrated recently with different quadruplex-specific antibodies. Despite the significant interest in these immunodetection approaches, dynamic detection of quadruplex in live cells remains elusive. Here, we report on NaphthoTASQ (N-TASQ), a next-generation quadruplex ligand that acts as a multiphoton turn-on fluorescent probe. Single-step incubation of human and mouse cells with N-TASQ enables the direct detection of RNA-quadruplexes in untreated cells (no fixation, permeabilization or mounting steps), thus offering a unique, unbiased visualization of quadruplexes in live cells.

Damage in Escherichia coli Cells Treated with a Combination of High Hydrostatic Pressure and Subzero Temperature

ABSTRACT The relationship between membrane permeability, changes in ultrastructure, and inactivation in Escherichia coli strain K-12TG1 cells subjected to high hydrostatic pressure treatment at room and subzero temperatures was studied. Propidium iodide staining performed before and after pressure treatment made it possible to distinguish between reversible and irreversible pressure-mediated cell membrane permeabilization. Changes in cell ultrastructure were studied using transmission electron microscopy (TEM), which showed noticeable condensation of nucleoids and aggregation of cytosolic proteins in cells fixed after decompression. A novel technique used to mix fixation reagents with the cell suspension in situ under high hydrostatic pressure (HHP) and subzero-temperature conditions made it possible to show the partial reversibility of pressure-induced nucleoid condensation. However, based on visual examination of TEM micrographs, protein aggregation did not seem to be reversible. Reversible cell membrane permeabilization was noticeable, particularly for HHP treatments at subzero temperature. A correlation between membrane permeabilization and cell inactivation was established, suggesting different mechanisms at room and subzero temperatures. We propose that the inactivation of E. coli cells under combined HHP and subzero temperature occurs mainly during their transiently permeabilized state, whereas HHP inactivation at room temperature is related to a balance of transient and permanent permeabilization. The correlation between TEM results and cell inactivation was not absolute. Further work is required to elucidate the effects of pressure-induced damage on nucleoids and proteins during cell inactivation.

Synergistic and antagonistic effects of combined subzero temperature and high pressure on inactivation of Escherichia coli.

ABSTRACT The combined effects of subzero temperature and high pressure on the inactivation of Escherichia coli K12TG1 were investigated. Cells of this bacterial strain were exposed to high pressure (50 to 450 MPa, 10-min holding time) at two temperatures (−20°C without freezing and 25°C) and three water activity levels (aw) (0.850, 0.992, and ca. 1.000) achieved with the addition of glycerol. There was a synergistic interaction between subzero temperature and high pressure in their effects on microbial inactivation. Indeed, to achieve the same inactivation rate, the pressures required at −20°C (in the liquid state) were more than 100 MPa less than those required at 25°C, at pressures in the range of 100 to 300 MPa with an aw of 0.992. However, at pressures greater than 300 MPa, this trend was reversed, and subzero temperature counteracted the inactivation effect of pressure. When the amount of water in the bacterial suspension was increased, the synergistic effect was enhanced. Conversely, when the aw was decreased by the addition of solute to the bacterial suspension, the baroprotective effect of subzero temperature increased sharply. These results support the argument that water compression is involved in the antimicrobial effect of high pressure. From a thermodynamic point of view, the mechanical energy transferred to the cell during the pressure treatment can be characterized by the change in volume of the system. The amount of mechanical energy transferred to the cell system is strongly related to cell compressibility, which depends on the water quantity in the cytoplasm.

Distributions of the growth rate of the germ tubes and germination time of Penicillium chrysogenum conidia depend on water activity

The effects of water activities for sporulation (a(wsp)) and germination (a(wge)) on the distributions of the growth rate of the germ tubes (mu) and the germination time (t(G)) of Penicillium chrysogenum conidia were determined by monitoring the length of the same germ tubes throughout the experiments automatically. No relationship between the individual t(G)s and mus could be established. Irrespective of the water activity for germination, mu was greater and t(G) was less for conidia produced at 0.95a(wsp) than that at 0.99a(wsp). At 0.99 a(wge) the mean and the standard deviation of t(G) were smaller than those obtained at 0.95a(wge). At 0.99a(wge), normal distributions for mu and t(G) were exhibited, but not at 0.95a(wge). The cumulative frequencies were used to reconstruct the germination curves. Great differences in the percentage of spores capable of germination (P(G)) and in the mean germination times between conidia produced at 0.95a(wsp) and at 0.99a(wsp) were clearly exhibited at 0.95a(wge), thus demonstrating the paramount influence of sporulation conditions on germination kinetics.

Impact of water activity of diverse media on spore germination of Aspergillus and Penicillium species

The effects of water activity (a(w)) of diverse media i/ culture medium for sporogenesis, a(w sp) ii/ liquid spore suspension medium, a(w su) and iii/ medium for germination, a(w ge), on the germination time t(G) of Aspergillus carbonarius, Aspergillus flavus, Penicillium chrysogenum and Penicillium expansum were assessed according to a screening matrix at 0.95 and 0.99 a(w). It was shown that i/ reduced t(G)s were obtained at 0.95 a(w sp) except for P. expansum ii/ a significant effect of a(w su) on t(G) was demonstrated for A. carbonarius, P. chrysogenum and P. expansum iii/ the most important factor for controlling the germination time was the medium for germination except for A. carbonarius (a(w su)). In accordance with the fact that fungal spores can swell as soon as they are suspended in an aqueous solution it is recommended to re-suspend fungal spores in a solution at the same water activity as that of subsequent germination studies.

Direct investigation of viscosity of an atypical inner membrane of Bacillus spores: A molecular rotor/FLIM study

We utilize the fluorescent molecular rotor Bodipy-C12 to investigate the viscoelastic properties of hydrophobic layers of bacterial spores Bacillus subtilis. The molecular rotor shows a marked increase in fluorescence lifetime, from 0.3 to 4ns, upon viscosity increase from 1 to 1500cP and can be incorporated into the hydrophobic layers within the spores from dormant state through to germination. We use fluorescence lifetime imaging microscopy to visualize the viscosity inside different compartments of the bacterial spore in order to investigate the inner membrane and relate its compaction to the extreme resistance observed during exposure of spores to toxic chemicals. We demonstrate that the bacterial spores possess an inner membrane that is characterized by a very high viscosity, exceeding 1000cP, where the lipid bilayer is likely in a gel state. We also show that this membrane evolves during germination to reach a viscosity value close to that of a vegetative cell membrane, ca. 600cP. The present study demonstrates quantitative imaging of the microscopic viscosity in hydrophobic layers of bacterial spores Bacillus subtilis and shows the potential for further investigation of spore membranes under environmental stress.

Absolute humidity influences the seasonal persistence and infectivity of human norovirus

ABSTRACT Norovirus (NoV) is one of the main causative agents of acute gastroenteritis worldwide. In temperate climates, outbreaks peak during the winter season. The mechanism by which climatic factors influence the occurrence of NoV outbreaks is unknown. We hypothesized that humidity is linked to NoV seasonality. Human NoV is not cultivatable, so we used cultivatable murine norovirus (MNV) as a surrogate to study its persistence when exposed to various levels of relative humidity (RH) from low (10% RH) to saturated (100% RH) conditions at 9 and 25°C. In addition, we conducted similar experiments with virus-like particles (VLPs) from the predominant GII-4 norovirus and studied changes in binding patterns to A, B, and O group carbohydrates that might reflect capsid alterations. The responses of MNV and VLP to humidity were somewhat similar, with 10 and 100% RH exhibiting a strong conserving effect for both models, whereas 50% RH was detrimental for MNV infectivity and VLP binding capacity. The data analysis suggested that absolute humidity (AH) rather than RH is the critical factor for keeping NoV infectious, with an AH below 0.007 kg water/kg air being favorable to NoV survival. Retrospective surveys of the meteorological data in Paris for the last 14 years showed that AH average values have almost always been below 0.007 kg water/kg air during the winter (i.e., 0.0046 ± 0.0014 kg water/kg air), and this finding supports the fact that low AH provides an ideal condition for NoV persistence and transmission during cold months.

Germination and inactivation of Bacillus subtilis spores induced by moderate hydrostatic pressure

In this study, we investigated the mechanisms of spore inactivation by high pressure at moderate temperatures to optimize the sterilization efficiency of high‐pressure treatments. Bacillus subtilis spores were first subjected to different pressure treatments ranging from 90 to 550 MPa at 40°C, with holding times from 10 min to 4 h. These treatments alone caused slight inactivation, which was related to the pressure‐induced germination of the spores. After these pressures treatments, the sensitivity of these processed spores to heat (80°C/10 min) or to high pressure (350 MPa/40°C/10 min) was tested to determine the pressure‐induced germination rate and the advancement of the spores in the germination process. The subsequent heat or pressure treatments were applied immediately after decompression from the first pressure treatment or after a holding time at atmospheric pressure. As already known, the spore germination is more efficient at low pressure level than at high pressure level. Our results show that this low germination efficiency at high pressure seemed not to be related either to a lower induction or a difference in the induction mechanisms but rather to an inhibition of enzyme activities which are involved in germination process. In fact, high pressure was necessary and very efficient in inducing spore germination. However, it seemed to slow the enzymatic digestion of the cortex, which is required for germinated spores to be inactivated by pressure. Although these results indicate that high‐pressure treatments are more efficient when the two treatments are combined, a small spore population still remained dormant and was not inactivated with any holding time or pressure level. Biotechnol. Bioeng. 2010;107: 876–883.

Cell inactivation and membrane damage after long-term treatments at sub-zero temperature in the supercooled and frozen states

The survival of cells subjected to cooling at sub‐zero temperature is of paramount concern in cryobiology. The susceptibility of cells to cryopreservation processes, especially freeze‐thawing, stimulated considerable interest in better understanding the mechanisms leading to cell injury and inactivation. In this study, we assessed the viability of cells subjected to cold stress, through long‐term supercooling experiments, versus freeze‐thawing stress. The viability of Escherichia coli, Saccharomyces cerevisiae, and leukemia cells were assessed over time. Supercooled conditions were maintained for 71 days at −10°C, and for 4 h at −15°C, and −20°C, without additives or emulsification. Results showed that cells could be inactivated by the only action of sub‐zero temperature, that is, without any water crystallization. The loss of cell viability upon exposure to sub‐zero temperatures is suggested to be caused by exposure to cold shock which induced membrane damage. During holding time in the supercooled state, elevated membrane permeability results in uncontrolled mass transfer to and from the cell maintained at cold conditions and thus leads to a loss of viability. With water crystallization, cells shrink suddenly and thus are exposed to cold osmotic shock, which is suggested to induce abrupt loss of cell viability. During holding time in the frozen state, cells remain suspended in the residual unfrozen fraction of the liquid and are exposed to cold stress that would cause membrane damage and loss of viability over time. However, the severity of such a stress seems to be moderated by the cell type and the increased solute concentration in the unfrozen fraction of the cell suspension. Biotechnol. Bioeng.

Modification of plasma membrane organization in tobacco cells elicited by cryptogein.

The ordering and organization of the plasma membrane of tobacco suspension cells as well as membrane fluidity, is modified during the early steps of the signaling cascade induced by an elicitor of defense reactions. Lipid mixtures within artificial membranes undergo a separation into liquid-disordered and liquid-ordered phases. However, the existence of this segregation into microscopic liquid-ordered phases has been difficult to prove in living cells, and the precise organization of the plasma membrane into such phases has not been elucidated in plant cells. We developed a multispectral confocal microscopy approach to generate ratiometric images of the plasma membrane surface of Bright Yellow 2 tobacco (Nicotiana tabacum) suspension cells labeled with an environment sensitive fluorescent probe. This allowed the in vivo characterization of the global level of order of this membrane, by which we could demonstrate that an increase in its proportion of ordered phases transiently occurred in the early steps of the signaling triggered by cryptogein and flagellin, two elicitors of plant defense reactions. The use of fluorescence recovery after photobleaching revealed an increase in plasma membrane fluidity induced by cryptogein, but not by flagellin. Moreover, we characterized the spatial distribution of liquid-ordered phases on the membrane of living plant cells and monitored their variations induced by cryptogein elicitation. We analyze these results in the context of plant defense signaling, discuss their meaning within the framework of the “membrane raft” hypothesis, and propose a new mechanism of signaling platform formation in response to elicitor treatment.