Fluorescent biomarkers are widely used in cell biology to study gene expression and protein localization. Translational fusions, where a fluorescent protein is directly linked to a protein of interest, allow researchers to monitor subcellular distribution, while transcriptional fusions are used to assess promoter activity. Despite their extensive application, the potential physiological impact of these fluorescent tags on host cells remains largely overlooked. In this study, we investigated how the Green Fluorescent Protein (GFP) influences stress responses in the yeast Saccharomyces cerevisiae. We generated translational fusions of GFP with two proteins: Pab1p, a key component of stress granules, and Sur7p, a membrane-associated protein involved in the organization of Can1-enriched plasma membrane domains. These targets were selected because the cellular behavior of S. cerevisiae under varying heat and oxidative stress conditions remains incompletely understood. Our main findings indicate that the Pab1p-GFP fusion confers increased resistance to heat shock compared to the wild-type strain. Furthermore, strains expressing GFP-tagged proteins displayed altered cultivability under oxidative stress, suggesting that the presence of GFP can modulate the cellular stress response. In silico structural analysis confirmed that GFP fusion does not alter the overall 3D structure or function of the tagged proteins. This suggests that the observed phenotypic differences are likely due to the intrinsic properties of GFP, particularly its known ability to scavenge reactive oxygen species (ROS). These results highlight an important consideration for researchers using fluorescent tags: while GFP is generally considered a neutral reporter, it can influence cellular behaviour under stress, potentially affecting the interpretation of experimental outcomes.

Experiments exposing Saccharomyces cerevisiae to glucose limitation (calorie restriction) are widely used to determine impacts on cell health as a model for aging. Using growth on plates and in liquid culture, we demonstrated that calorie restriction reduces fitness in subsequent nutrient-limited environments. Yeast grown in a calorie-restricted environment took longer to emerge from the lag phase, had an extended doubling time and had a lower percentage of culturability. Cells grown under moderate calorie restriction were able to withstand a gradual heat stress in a similar manner to cells grown without calorie restriction but fared less well with a sudden heat shock. Yeast grown under extreme calorie restriction were less fit when exposed to gradual heating or heat shock. Using RNAseq analysis, we provide novel insight into the mechanisms underlying this response, showing that in the absence of calorie restriction, genes whose products are involved in energy metabolism (glycolysis/gluconeogenesis and the citrate cycle) are predominantly overexpressed when yeasts were exposed to gradual heating, whereas this was not the case when they were exposed to shock. We show that both the culture history and the current environment must be considered when assaying physiological responses, and this has wider implications when developing strategies for the propagation, preservation or destruction of microbial cells.

The current study investigated the impact of Saccharomyces cerevisiae fermentation on apricot kernel oil extraction and the resulting quality characteristics of the oil. The seed kernels underwent physicochemical and antioxidant studies after being separated, gelatinized, and fermented. The results revealed 43.6% of oil yield by fermentation. The extracted oil had a lower acid value (3.74mg KOH/g), and peroxide value (4.25meq/kg). The investigation of additional oil properties was included, like iodine value (100.26mg KOH/g), saponification value (190.5mg KOH/g), specific gravity (0.919), viscosity (4.62), moisture content (0.592%), and refractive index (1.4836), which signifies optimum physicochemical parameters of oil. As per the FTIR data, some distinct functional groups like C-H stretch and C=O, C=C, CH3, OH, and R-O-R esters were found in the extracted oil. The oil's fatty acid composition, with 50.47% saturated, 22.23% mono-unsaturated, and 35.13% polyunsaturated fatty acids, enhances stability, oxidative resistance, and nutritional value, justifying its favorable physicochemical properties. The DPPH radical scavenging activity of the oil showed to have good antioxidant activity. Metabolic modeling of S. cerevisiae provided information about compounds received after fermentation. Hence, it can be concluded that fermentation is a suitable approach to extract apricot seed kernel oil with beneficial properties.

Oenococcus oeni is the main lactic acid bacterium associated with malolactic fermentation (MLF) of wines. MLF plays an important role in determining the final quality of wines. Nevertheless, due to the stressful conditions inherent to wine and especially acidity, MLF may be delayed. This study aimed to explore by adaptive evolution improvements in the acid tolerance of starters but also to gain a better understanding of the mechanisms involved in adaptation toward acidity. Four independent populations of the O. oeni ATCC BAA-1163 strain were propagated (approximately 560 generations) in a temporally varying environment, consisting in a gradual pH decrease from pH 5.3 to pH 2.9. Whole genome sequence comparison of these populations revealed that more than 45% of the substituted mutations occurred in only five loci for the evolved populations. One of these five fixed mutations affects mae, the first gene of the citrate operon. When grown in an acidic medium supplemented with citrate, a significantly higher bacterial biomass was produced with the evolved populations compared to the parental strain. Furthermore, the evolved populations slowed down their citrate consumption at low pH without impacting malolactic performance.

Human noroviruses (HuNoVs) are the predominant etiological agent of viral gastroenteritis in all age groups worldwide. Mutations over the years have affected noroviruses' responses to environmental conditions due to the arrangement of amino acid residues exposed on the VP1 capsid surface of each strain. The GII.4 HuNoV genotype has been the predominant variant for decades, while the GII.17 genotype has often been detected in East Asia since 2014. Here, GII.17 and GII.4 baculovirus-expressed VLPs (virus-like particles) were used to study the biological (binding to HuNoV ligand, namely the ABO and Lewis antigens) and physicochemical properties (size, morphology, and charge) of the HuNoV capsid under different conditions (temperature, pH, and ionic strength). GII.17 showed stability at low and high ionic strength, while GII.4 aggregated at an ionic strength of 10 mM. The nature of the buffers influences the morphology and stability of the VLPs. Here, both VLPs were highly stable from pH 7-8.5 at 25 degrees C. VLPs retained HBGA binding capability for the pH, ionic strength and temperature encountered in the stomach (fed state) and the small intestine. Increasing the temperature to above 65 degrees C altered the morphology of VLPs, causing aggregation, and decreased their affinity to HBGAs. Comparing both isolates, GII.17 showed a better stability profile and higher affinity to HBGAs than GII.4, making them interesting candidate particles for a future norovirus vaccine. Biological and physicochemical studies of VLPs are as perti-nent as ever in view of the future arrival of VLP-based HuNoV vaccines.

Although fluorescent proteins are widely used as biomarkers (Yin), no study focuses on their influence on the microbial stress response. Here, the Green Fluorescent Protein (GFP) was fused to two proteins of interest in Saccharomyces cerevisiae. Pab1p and Sur7p, respectively involved in stress granules structure and in Can1 membrane domains. These were chosen since questions remain regarding the understanding of the behavior of S. cerevisiae facing different heat kinetics or oxidative stresses. The main results showed that Pab1p-GFP fluorescent mutant displayed a higher resistance than that of the wild type under a heat shock. Moreover, fluorescent mutants exposed to oxidative stresses displayed changes in the cultivability compared to the wild type strain. In silico approaches showed that the presence of the GFP did not influence the structure and so the functionality of the tagged proteins meaning that changes in yeast resistance were certainly related to GFP ROS-scavenging ability (Yang).

Mercury (Hg) pollution is a global issue due to the high toxicity and wide dispersion of Hg around the world. Whether due to anthropogenic activities or natural processes, Hg emissions are steadily increasing, with very high levels in some regions, directly threatening human and ecosystem health. However, bacteria and fungi have evolved and adapted in response to Hg-induced stress and have developed tolerance mechanisms, notably based on the mer operon system that is involved in Hg uptake and biovolatilization via Hg reduction reactions. Other processes, such as bioaccumulation or extracellular sequestration, are involved in Hg resistance, and the study of contaminated soils has allowed the isolation of a number of microorganisms capable of these mechanisms, with strong potential for the implementation of bioremediation approaches. In addition to playing an important role in determining the fate of Hg in the biogeochemical cycle, these microorganisms can indeed be applied to reduce Hg concentrations or at least stabilize Hg for the remediation of polluted soils. Moreover, thanks to the development of biotechnological tools, bioremediation based on Hg-tolerant microorganisms can be optimized. Finally, these microorganisms are relevant candidates for biomonitoring, for example, through the engineering of biosensors, because the detection of Hg is a major issue in preserving the health of living beings.

Although relative air humidity (RH) strongly influences microbial survival, its use for fighting surface pathogens in the food industry has been inadequately considered. We asked whether RH control could destroy Listeria monocytogenes EGDe by envelope damage. The impact of dehydration in phosphate-buffered saline (PBS) at 75%, 68%, 43% and 11% RH on the bacterial envelope was investigated using flow cytometry and atomic force microscopy. Changes after rehydration in the protein secondary structure and peptidoglycan were investigated by infrared spectroscopy. Complementary cultivability measurements were performed by running dehydration–rehydration with combinations of NaCl (3–0.01%), distilled water, city water and PBS. The main results show that cell membrane permeability and cell envelope were greatly altered during dehydration in PBS at 68% RH followed by rapid rehydration. This damage led cells to recover only 67% of their initial volume after rehydration. Moreover, the most efficient way to destroy cells was dehydration and rehydration in city water. Our study indicates that rehydration of dried, sullied foods on surfaces may improve current cleaning procedures in the food industry.

Summary Although mechanisms involved in response of Saccharomyces cerevisiae to osmotic challenge are well described for low and sudden stresses, little is known about how cells respond to a gradual increase of the osmotic pressure (reduced water activity; aw) over several generations as it could encounter during drying in nature or in food processes. Using glycerol as a stressor, we propagated S. cerevisiae through a ramp of the osmotic pressure (up to high molar concentrations to achieve testing‐to‐destruction) at the rate of 1.5 MPa day‐1 from 1.38 to 58.5 MPa (0.990–0.635 aw). Cultivability (measured at 1.38 MPa and at the harvest osmotic pressure) and glucose consumption compared with the corresponding sudden stress showed that yeasts were able to grow until about 10.5 MPa (0.926 aw) and to survive until about 58.5 MPa, whereas glucose consumption occurred until 13.5 MPa (about 0.915 aw). Nevertheless, the ramp conferred an advantage since yeasts harvested at 10.5 and 34.5 MPa (0.778 aw) showed a greater cultivability than glycerol‐shocked cells after a subsequent shock at 200 MPa (0.234 aw) for 2 days. FTIR analysis revealed structural changes in wall and proteins in the range 1.38–10.5 MPa, which would be likely to be involved in the resistance at extreme osmotic pressure.

1-(4-Trimethylammoniophenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA‑DPH, Sigma Aldrich) was used to monitor physical properties of mitochondrial membranes of 7-to-8-mo-old mice. TMA‑DPH is composed of a cationic substitute (TMA) that anchors at the polar heads of the membrane, while allowing the fluorescent hydrophobic probe DPH to be located in nonpolar regions. To limit fluorescent noise, mitoplasts were resuspended in the Hypotonic Buffer (sucrose 9 mM, mannitol 29 mM, Hepes 0.3 mM, pH 7.4) as described hereafter. Experiments were repeated five times using independent samples, each from different mice. Decay-fluorescence measurements were performed on each sample at 37 °C after a measurement of the instrumental response of the spectrofluorimeter used to measure fluorescence-decay (“prompt”). Thirty µl of sample (DO600 = 0.055) diluted in 2.97 ml of Hypotonic Buffer were introduced into spectroscopic quartz cuvette with an optical path length of one cm (VWR International). Fluorescence-decay was measured by the time-correlated single-photon counting (TCSPC) method using a Horiba-Fluoromax-4® spectrofluorimeter (Horiba) equipped with a 370-nm laser diode (NanoLED C2, Horiba) as the source of excitation. Fluorescence decays were measured in TCSPC setup (Deltahub, Horiba). The Instrument Response Function (IRF) was about 160 ps (measured at 370 nm using the hypotonic buffer). Emission and excitation wavelength of TMA-DPH were respectively fixed at 370 nm and 431 ± 1.1 nm. Each decay curve corresponded to 10,000 counts.

In the present paper, the Layer by Layer (LbL) method using β-lactoglobulin and sodium alginate was performed to individually encapsulate Saccharomyces cerevisiae cells in microorganized shells in order to protect them against stresses during dehydration. Higher survival (∼1 log) for encapsulated yeast cells was effectively observed after air dehydration at 45°C. For the first time, the potentiality of Synchrotron-Fourier Transform InfraRed microspectroscopy (S-FTIR) was used at the single-cell level in order to analyze the contribution of the biochemical composition of non-encapsulated vs. encapsulated cells in response to dehydration. The microspectroscopy measurements clearly differentiated between non-encapsulated and encapsulated yeast cells in the amide band region. In the spectral region specific to lipids, the S-FTIR results indicated probably the decrease in membrane fluidity of yeast after dehydration without significant distinction between the two samples. These data suggested minor apparent chemical changes in cell attributable to the LbL system upon dehydration. More insights are expected regarding the lower mortality among encapsulated cells. Indeed the hypothesis that the biopolymeric layers could induce less damage in cell by affecting the transfer kinetics during dehydration-rehydration cycle, should be verified in further work.

In the context of microbiology, recent studies show the importance of ribonucleo-protein aggregates (RNPs) for the understanding of mechanisms involved in cell responses to specific environmental conditions. The assembly and disassembly of aggregates is a dynamic process, the characterization of the stage of their evolution can be performed by the evaluation of their number. The aim of this study is to propose a method to automatically determine the count of RNPs. We show that the determination of a precise count is an issue by itself and hence, we propose three textural approaches: a classical point of view using Haralick features, a frequency point of view with generalized Fourier descriptors, and a structural point of view with Zernike moment descriptors (ZMD). These parameters are then used as inputs for a supervised classification in order to determine the most relevant. An experiment using a specific Saccharomyces cerevisiae strain presenting a fusion between a protein found in RNPs (PAB1) and the green fluorescent protein was performed to benchmark this approach. The fluorescence was observed with two-photon fluorescence microscopy. Results show that the textural approach, by mixing ZMD with Haralick features, allows for the characterization of the number of RNPs.

Because of the ability of foodborne pathogens to survive in low-moisture foods, their decontamination is an important issue in food protection. This study aimed to clarify some of the cellular mechanisms involved in inactivation of foodborne pathogens after drying and subsequent heating. Individual strains of Salmonella Typhimurium, Salmonella Senftenberg, and Cronobacter sakazakii were mixed into whole milk powder and dried to different water activity levels (0.25 and 0.58); the number of surviving cells was determined after drying and subsequent thermal treatments in closed vessels at 90 and 100°C, for 30 and 120 s. For each condition, the percentage of unculturable cells was estimated and, in parallel, membrane permeability and respiratory activity were estimated by flow cytometry using fluorescent probes. After drying, it was clearly observable that the percentage of unculturable cells was correlated with the percentage of permeabilized cells (responsible for 20–40% of the total inactivated bacteria after drying), and to a lesser degree with the percentage of cells presenting with loss of respiratory activity. In contrast, the percentages of unculturable cells observed after heat treatment were strongly correlated with the loss of respiratory activity and weakly with membrane permeability (for 70–80% of the total inactivated bacteria after heat treatment). We conclude that cell inactivation during drying is closely linked to membrane permeabilization and that heat treatment of dried cells affects principally their respiratory activity. These results legitimize the use of time–temperature scales and allow better understanding of the cellular mechanisms of bacterial death during drying and subsequent heat treatment. These results may also allow better optimization of the decontamination process to ensure food safety by targeting the most deleterious conditions for bacterial cells without denaturing the food product.

Salmonella Typhimurium and Cronobacter sakazakii are two foodborne pathogens involved in neonatal infections from milk powder and infant formula. Their ability to survive in low-moisture food and during processing from the decontamination to the dried state is a major issue in food protection. In this work, we studied the effects of the drying process on Salmonella Typhimurium and Cronobacter sakazakii, with the aim of identifying the drying parameters that could promote greater inactivation of these two foodborne pathogens. These two bacteria were dried under different atmospheric relative humidities in milk and phosphate-buffered saline, and the delays in growth recovery and cultivability were followed. We found that water activity was related to microorganism resistance. C. sakazakii was more resistant to drying than was S. Typhimurium, and milk increased the cultivability and recovery of these two species. High drying rates and low final water activity levels (0.11-0.58) had a strong negative effect on the growth recovery and cultivability of these species. In conclusion, we suggest that effective use of drying processes may provide a complementary tool for food decontamination and food safety during the production of low-moisture foods. (C) 2016 Elsevier Ltd. All rights reserved.

Increased levels of 7-ketocholesterol (7KC), which results mainly from cholesterol auto-oxidation, are often found in the plasma and/or cerebrospinal fluid of patients with neurodegenerative diseases and might contribute to activation of microglial cells involved in neurodegeneration. As major cellular dysfunctions are induced by 7KC, it is important to identify molecules able to impair its side effects. Since consumption of olive and argan oils, and fish is important in the Mediterranean diet, the aim of the study was to determine the ability of oleic acid (OA), a major compound of olive and argan oil, and docosahexaenoic acid (DHA) present in fatty fishes, such as sardines, to attenuate 7KC-induced cytotoxic effects. Since elaidic acid (EA), the trans isomer of OA, can be found in hydrogenated cooking oils and fried foods, its effects on 7KC-induced cytotoxicity were also determined. In murine microglial BV-2 cells, 7KC induces cell growth inhibition, mitochondrial dysfunctions, reactive oxygen species overproduction and lipid peroxidation, increased plasma membrane permeability and fluidity, nuclei condensation and/or fragmentation and caspase-3 activation, which are apoptotic characteristics, and an increased LC3-II/LC3-I ratio, which is a criterion of autophagy. 7KC is therefore a potent inducer of oxiapoptophagy (OXIdation + APOPTOsis + autoPHAGY) on BV-2 cells. OA and EA, but not DHA, also favor the accumulation of lipid droplets revealed with Masson’s trichrome, Oil Red O, and Nile Red staining. The cytotoxicity of 7KC was strongly attenuated by OA and DHA. Protective effects were also observed with EA. However, 7KC-induced caspase-3 activation was less attenuated with EA. Different effects of OA and EA on autophagy were also observed. In addition, EA (but not OA) increased plasma membrane fluidity, and only OA (but not EA) was able to prevent the 7KC-induced increase in plasma membrane fluidity. Thus, in BV-2 microglial cells, the principal fatty acids of the Mediterranean diet (OA, DHA) were able to attenuate the major toxic effects of 7KC, thus reinforcing the interest of natural compounds present in the Mediterranean diet to prevent the development of neurodegenerative diseases.

This study was carried out in order to develop experimental methodology using a camera to monitor the evolution of the surface of a liquid droplet deposited on a solid surface composed of polypropylene. The droplet was exposed to various ambient relative humidity conditions (113%, 43.2%, 68.9% and 75.5%). Two types of liquid were investigated: distilled water and water containing nutritive substances (salmon "juice"). At 11.3% relative "humidity, it takes 40% longer to evaporate a water droplet (initial weight 0.36 g, volume 360 mu L, radius 6.5 x. 10(-3) m) than a salmon "juice" droplet (3.66 h for distilled water, 2.83 h for salmon "juice"). In the case of the distilled water droplet, the wet surface decreases gradually and completely disappears at the end of the process. In the case of the salmon "juice" droplet, the wet surface is constant for about 2 h and then decreases gradually because of drying from the edge towards the center of the droplet. A simple equation making it possible to predict the drying rate as a function of air humidity was developed. Also, measurements of the loss of cultivability of Listeria monocytogenes under different relative humidity conditions were carried out experimentally. The relationship between the relative humidity, droplet drying time and loss of cultivability was analyzed. It was observed that for 113%, 43.2% and 68.9% relative humidity conditions, the drying time and the loss of cultivability can be correlated, while at 75.5% relative humidity, the phenomena are more complex. This study shows that the relative humidity of air can potentially be controlled in order to limit bacterial growth, thus enhancing hygiene in food plants. (C) 2017 Elsevier Ltd. All rights reserved.

[Technical Session 1 – Intervention Strategies] Introduction: Salmonella enterica and Cronobacter sakazakii are foodborne pathogens responsible for severe infant illness. Their ability to survive in harsh environmental conditions make these species a matter of concern for the low moisture food industry. Purpose: This study aimed to evaluate and understand the impact of drying conditions on survival, physiology, and invasion capacity of Salmonella Typhimurium and C. sakazakii.Methods: Salmonella Typhimurium and C. sakazakii were mixed into whole milk powder and dried at different water activity (aw) levels (0.25, 0.58 and 0.80). For each strain, the impact of each drying condition was evaluated by estimating the loss of cultivability, membrane permeabilization, and the loss of a respiratory enzymatic activity by flow cytometry. The invasion capacity in Caco-2 cells was, also, evaluated after drying for each bacterium by the gentamicin test.Results: Our results showed that intermediary initial drying kinetics increased bacterial inactivation. No significant differences were observed between bacterial cultivability at aw 0.25 and 0.58. Nevertheless, the bacterial cultivability was significantly higher (p <0.05) at aw 0.80 than at 0.25 or 0.58. An increase in percentages of uncultivable cells correlated with percentages of permeabilized cells. Furthermore, our results showed that drying (at 0.80, 0.58, and 0.25) significantly increased (p <0.05) the invasion capacity of S. Typhimurium and C. sakazakii.Significance: These results indicate that drying parameters have a high impact on S. enterica and C. sakazakii and could be managed to promote foodborne pathogen inactivation. Drying could, also, be improved to avoid virulence pathway activation and ensure the safety of dried food products.