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Since the growing number of fungi resistant to the fungicides used is becoming a serious threat to human health, animals, and crops, there is a need to find other effective approaches in the eco-friendly suppression of fungal growth. One of the main mechanisms of the development of resistance in fungi, as well as in bacteria, to antimicrobial agents is quorum sensing (QS), in which various lactone-containing compounds participate as signaling molecules. This work aimed to study the effectiveness of action of enzymes exhibiting lactonase activity against fungal signaling molecules. For this, the molecular docking method was used to estimate the interactions between these enzymes and different lactone-containing QS molecules of fungi. The catalytic characteristics of enzymes such as lactonase AiiA, metallo-β-lactamase NDM-1, and organophosphate hydrolase His6-OPH, selected for wet experiments based on the results of computational modeling, were investigated. QS lactone-containing molecules (butyrolactone I and γ-heptalactone) were involved in the experiments as substrates. Further, the antifungal activity of the enzymes was evaluated against various fungal and yeast cells using bioluminescent ATP-metry. The efficient hydrolysis of γ-heptalactone by all three enzymes and butyrolactone I by His6-OPH was demonstrated for the first time. The high antifungal efficacy of action of AiiA and NDM-1 against most of the tested fungal cells was revealed. [ABSTRACT FROM AUTHOR]

Since the growing number of fungi resistant to the fungicides used is becoming a serious threat to human health, animals, and crops, there is a need to find other effective approaches in the eco-friendly suppression of fungal growth. One of the main mechanisms of the development of resistance in fungi, as well as in bacteria, to antimicrobial agents is quorum sensing (QS), in which various lactone-containing compounds participate as signaling molecules. This work aimed to study the effectiveness of action of enzymes exhibiting lactonase activity against fungal signaling molecules. For this, the molecular docking method was used to estimate the interactions between these enzymes and different lactone-containing QS molecules of fungi. The catalytic characteristics of enzymes such as lactonase AiiA, metallo-β-lactamase NDM-1, and organophosphate hydrolase His6-OPH, selected for wet experiments based on the results of computational modeling, were investigated. QS lactone-containing molecules (butyrolactone I and γ-heptalactone) were involved in the experiments as substrates. Further, the antifungal activity of the enzymes was evaluated against various fungal and yeast cells using bioluminescent ATP-metry. The efficient hydrolysis of γ-heptalactone by all three enzymes and butyrolactone I by His6-OPH was demonstrated for the first time. The high antifungal efficacy of action of AiiA and NDM-1 against most of the tested fungal cells was revealed.

Industrial fermentation processes strive for high robustness to ensure optimal and consistent performance. Medium components, fermentation products, and physical perturbations may cause stress and lower performance. Cellular stress elicits a range of responses, whose extracellular manifestations have been extensively studied; whereas intracellular aspects remain poorly known due to lack of tools for real-time monitoring. Genetically encoded biosensors have emerged as promising tools and have been used to improve microbial productivity and tolerance toward industrially relevant stresses. Here, fluorescent biosensors able to sense the yeast intracellular environment (pH, ATP levels, oxidative stress, glycolytic flux, and ribosome production) were implemented into a versatile and easy-to-use toolbox. Marker-free and efficient genome integration at a conserved site on chromosome X of Saccharomyces cerevisiae strains and a commercial Saccharomyces boulardii strain was developed. Moreover, multiple biosensors were used to simultaneously monitor different intracellular parameters in a single cell. Even when combined together, the biosensors did not significantly affect key physiological parameters, such as specific growth rate and product yields. Activation and response of each biosensor and their interconnection were assessed using an advanced micro-cultivation system. Finally, the toolbox was used to screen cell behavior in a synthetic lignocellulosic hydrolysate that mimicked harsh industrial substrates, revealing differences in the oxidative stress response between laboratory (CEN.PK113-7D) and industrial (Ethanol Red) S. cerevisiae strains. In summary, the toolbox will allow both the exploration of yeast diversity and physiological responses in natural and complex industrial conditions, as well as the possibility to monitor production processes. [ABSTRACT FROM AUTHOR]

Industrial fermentation processes strive for high robustness to ensure optimal and consistent performance. Medium components, fermentation products, and physical perturbations may cause stress and lower performance. Cellular stress elicits a range of responses, whose extracellular manifestations have been extensively studied; whereas intracellular aspects remain poorly known due to lack of tools for real-time monitoring. Genetically encoded biosensors have emerged as promising tools and have been used to improve microbial productivity and tolerance toward industrially relevant stresses. Here, fluorescent biosensors able to sense the yeast intracellular environment (pH, ATP levels, oxidative stress, glycolytic flux, and ribosome production) were implemented into a versatile and easy-to-use toolbox. Marker-free and efficient genome integration at a conserved site on chromosome X of Saccharomyces cerevisiae strains and a commercial Saccharomyces boulardii strain was developed. Moreover, multiple biosensors were used to simultaneously monitor different intracellular parameters in a single cell. Even when combined together, the biosensors did not significantly affect key physiological parameters, such as specific growth rate and product yields. Activation and response of each biosensor and their interconnection were assessed using an advanced micro-cultivation system. Finally, the toolbox was used to screen cell behavior in a synthetic lignocellulosic hydrolysate that mimicked harsh industrial substrates, revealing differences in the oxidative stress response between laboratory (CEN.PK113-7D) and industrial (Ethanol Red) S. cerevisiae strains. In summary, the toolbox will allow both the exploration of yeast diversity and physiological responses in natural and complex industrial conditions, as well as the possibility to monitor production processes.

The maintenance of intracellular processes, like organelle transport and cell division, depend on bidirectional movement along microtubules. These processes typically require kinesin and dynein motor proteins, which move with opposite directionality. Because both types of motors are often simultaneously bound to the cargo, regulatorymechanisms are required to ensure controlled directional transport. Recently, it has been shown that parameters like mechanical motor activation, ATP concentration and roadblocks on the microtubule surface differentially influence the activity of kinesin and dynein motors in distinct manners. However, how these parameters affect bidirectional transport systems has not been studied. Here, we investigate the regulatory influence of these three parameters using in vitro gliding motility assays and stochastic simulations. We find that the number of active kinesin and dynein motors determines the transport direction and velocity, but that variations in ATP concentration and roadblock density have no significant effect. Thus, factors influencing the force balance between opposite motors appear to be important, whereas the detailed stepping kinetics and bypassing capabilities of the motors only have a small effect. [ABSTRACT FROM AUTHOR]

Rainwater has been found to be a valuable source of drinking water in Europe, especially in such crisis situations as those caused by contamination of water uptake into water supply systems, large-scale floods or terrorist attacks (e.g., biological weapons). The microbiological quality of water plays a significant role, which is directly related to the potential health risks associated with harvested rainwater (including rainwater stored in the tanks). Microbial contamination is commonly found in rainwater. However, in the literature, detailed results of qualitative and quantitative microbiological assessments are sparse and remain unexplored. Therefore, the aim of this study was to investigate and analyze changes in the microbiological quality of roof-harvested rainwater stored in the tanks, depending on the collection conditions (type of roof surface), storage duration and season. Authors elucidate that conditions such as storage duration, the season in which rainwater is collected, the roof-like surface types and morphology of the catchment area highly affect rainwater quality. This study showed that rainwater harvested from a galvanized steel sheet roof had the best microbial quality, regarding the lowest number of bacteria, while rainwater from a flat roof covered with epoxy resin was the worst. Further, it was detected that rainwater collected in autumn and spring obtained the best microbiological quality. Moreover, a decrease in the number of bacteria was observed in correlation to storage duration. The water became sanitary safe after six weeks of storage at 12 °C. Its use for purposes requiring drinking water quality before six weeks of storage required disinfection.

Oocyte cytoplasmic maturation is critical in determining the success of subsequent embryo development. It has been proposed that light in the red-to-near infrared optical region (~600-1000 nm) enhances cellular metabolic activity through activation of the mitochondrial respiratory chain, increasing intracellular ATP concentrations, and has the potential to modify the oocyte metabolome. Previous research indicated a greater cleavage rate and similar blastocyst rates for bovine oocytes treated with photobiomodulation at hours 16 and 20 from the start of maturation compared with control. For the present study, two separate experiments were conducted to determine ATP concentration in oocytes during maturation after photobiomodulation treatment. A standardized in vitro maturation (IVM) protocol and commercial media (IVF Bioscience) were used. Briefly, bovine ovaries were obtained at a local slaughterhouse, cumulus oocyte complexes (COCs) were collected by aspiration of small (2 to 6 mm) follicles. For experiment 1, compact COCs were cultured in in vitro maturation media (n = 30/well) and treated with no light (control); light (660-665nm) for 10 minutes at hour 16 from start of maturation (L-16) and light for 10 minutes at hour 20 from the start of maturation (L-20). At hour 24 of in vitro maturation, COCs were vortexed to remove cumulus, stained with BioTracker ATP-Red Live Cell Dye (Sigma-Aldrich SCT045), imaged with Cytation-1 (Agilent Technologies Inc.) in 6 replicates. For experiment 2, COCs were cultured in in vitro maturation media (n = 40/well) and treated with no light (control) and light (660-665nm) for 10 minutes at hour 16 from start of maturation (L-16). At hours 18, 20, 22 and 24 of in vitro maturation, COCs were vortexed to remove cumulus, stained with BioTracker ATP-Red Live Cell Dye, and imaged with Cytation-1 in 4 replicates. For experiment 1, one-way ANOVA and Student's T-Test were performed (JMP software 16.0); in experiment 2, each well was considered as an experimental unit in a model of repeated measures, main effects of time and treatment, blocked by replicate (Mixed procedure, SAS Institute Inc.). In experiment 1, there was a significant (P < 0.05) effect of treatment; ATP concentrations were greater in L-16 than control, and L-20 was intermediate. ATP concentrations were 19,579.3a ± 596.5, 21,652.0b ± 602.7 and 19,710.9ab ± 570.6 relative fluorescence units (RFU) for control, L-16, and L-20 respectively. In experiment 2, there was a significant (P < 0.05) effect of treatment and no significant effect of time. Overall ATP concentrations were 25,668 ± 894.6 and 28,332 ± 894.6 RFU for control and L-16 respectively. In conclusion, photobiomodulation treatment with LED 660-665nm for 10 minutes at hour 16 from start of oocyte maturation increases intracellular ATP concentrations from hour 18 to 24 of maturation. [ABSTRACT FROM AUTHOR]

In vitro motility assay (IVMA) experiments were performed to analyze the movement of actin filaments sliding on a pavement of myosin molecules at different [ATP] and [ADP]. In standard experimental conditions at [ATP] = 2 mM, about 80% of the actin filaments move in unloaded conditions with a constant velocity. However, a fraction of at least 20% static actin filaments is always present. The accepted explanation is the occurrence of damaged 'rigor'-like myosin heads that do not undergo the normal ATP-dependent cycling motion. However, in a series of IVMA experiments performed at different [ATP] we observed that the mobility of actin filaments increased with lowering [ATP]. We investigated the influence of [ATP] on the number of mobile actin filaments. IVMA experiments were performed at controlled nucleotide concentrations and the percentage of mobile filaments accurately determined by specific operator-guided software. The value of Δ G involved was determined. Results showed that the number of mobile actin filaments sliding on type 2B heavy meromyosin isoform (2B HMM) increased at very low [ATP] accompanied by less negative Δ G values. Similar results were obtained by increasing [ADP]. Performing experiments at the same [ATP] with different myosin types, we found a higher number of mobile actin filaments on slow type 1 HMM with respect to type 2B HMM while the highest number of mobile actin filaments was found on single-head myosin (S1 fraction). We also found that [ATP] did not influence the percentage of mobile actin filaments sliding on S1. Our results reveal novel aspects of actomyosin interaction. [ABSTRACT FROM AUTHOR]

The homeostasing kinetics of one of the main parameters of the energy metabolism of erythrocytes, i.e., the pH value of their cytoplasm is analyzed. The balance curve of cytoplasm acidification and alkalization rates on the ATP concentration ( n)-cytoplasm pH plane is obtained. It is found that its construction requires the use of the balance equation of active and passive cation (K and Na) transport rate through cytoplasmic membranes of erythrocytes. These rates are considered as functions of pH and ATP concentrations in cytoplasm. The factors affecting the position of the obtained balance curve are determined. The assumption is made on the existence of special regulatory substances in erythrocytes,which can probably changemetabolic process rates. [ABSTRACT FROM AUTHOR]

Images of human erythrocytes from a healthy donor were recorded under differential interference contrast (DIC) microscopy; they were acquired rapidly (~336 Hz) and the intensity of the centermost pixel of each cell was recorded for ~60 s (20,000 values). Various techniques were used to analyze the data, including detrended fluctuation analysis (DFA) and multiscale entropy (MSE); however, power spectrum analysis was deemed the most appropriate for metrifying and comparing results. This analysis was used to compare cells from young and old populations, and after perturbing normal conditions, with changes in temperature, adenosine triphosphate (ATP) concentration (using NaF, an inhibitor of glycolysis, and α-toxin, a pore-forming molecule used to permeabilize red cells to ATP), and water transport rates [using glycerol, and p-chloromercuriphenylsulfonic acid (pCMBS) to inhibit aquaporins, AQPs]. There were measurable differences in the membrane fluctuation characteristics in populations of young and old cells, but there was no significant change in the flickering time series on changing the temperature of an individual cell, by depleting it of ATP, or by competing with the minor water exchange pathway via AQP3 using glycerol. However, pCMBS, which inhibits AQP1, the major water exchange pathway, inhibited flickering in all cells, and yet it was restored by the membrane intercalating species dibutyl phthalate (DBP). We developed a computer model to simulate acquired displacement spectral time courses and to evaluate various methods of data analysis, and showed how the flexibility of the membrane, as defined in the model, affects the flickering time course. [ABSTRACT FROM AUTHOR]

Due to the discrepancy between organ donors and receptors, the use of marginal livers (e.g., non-heart-beating-donor grafts) for transplantation purpose increased. The potential of a short-term aerobic machine perfusion (post-conditioning) for “less than optimal” grafts after cold storage (CS) was recently demonstrated. In our study, the optimal time course of post-conditioning (PC) is to be evaluated. Livers from male Wistar rats were withdrawn 30 min after cardiac arrest and flushed with histidine tryptophan ketoglutarate (HTK) solution. Then they were stored in HTK at 4°C for 18 h. After 16 h, some livers were put on PC by cold perfusion with HTK for 0.5, 1, 2 or 3 h. Afterwards, the viability of the organs was estimated by warm reperfusion (2 h) in vitro. After 1 h of PC, a significant increase in bile production and a decrease in enzyme release could be detected in comparison to CS. The adenosine triphosphate content of the PC livers after 1 h of treatment was significant higher than in CS organs. No markers for apoptosis could be detected after 1 h PC. It can be concluded that a PC of 1 h after cold storage can ameliorate the organ viability of marginal livers. The extension or abbreviation of PC time seems to have no further beneficial effects. [ABSTRACT FROM AUTHOR]

Rainwater has been found to be a valuable source of drinking water in Europe, especially in such crisis situations as those caused by contamination of water uptake into water supply systems, large-scale floods or terrorist attacks (e.g., biological weapons). The microbiological quality of water plays a significant role, which is directly related to the potential health risks associated with harvested rainwater (including rainwater stored in the tanks). Microbial contamination is commonly found in rainwater. However, in the literature, detailed results of qualitative and quantitative microbiological assessments are sparse and remain unexplored. Therefore, the aim of this study was to investigate and analyze changes in the microbiological quality of roof-harvested rainwater stored in the tanks, depending on the collection conditions (type of roof surface), storage duration and season. Authors elucidate that conditions such as storage duration, the season in which rainwater is collected, the roof-like surface types and morphology of the catchment area highly affect rainwater quality. This study showed that rainwater harvested from a galvanized steel sheet roof had the best microbial quality, regarding the lowest number of bacteria, while rainwater from a flat roof covered with epoxy resin was the worst. Further, it was detected that rainwater collected in autumn and spring obtained the best microbiological quality. Moreover, a decrease in the number of bacteria was observed in correlation to storage duration. The water became sanitary safe after six weeks of storage at 12 °C. Its use for purposes requiring drinking water quality before six weeks of storage required disinfection. [ABSTRACT FROM AUTHOR]

Endogenous elemental sulfur (S°) was measured in dormant α‐spores of Phomopsis viticola Sacc. (ATCC 44940) from young (25‐day‐old) and aging (105‐day‐old) cultures grown on malt extract agar medium enriched with [35S]‐MgSO4. Endogenous S° from the mitochondrial fraction, and the lipid and aqueous cytoplasmic fractions of young and aging α‐spores were purified by column chromatography followed by thin-layer chromatography. The purity of mitochondrial pellets were checked by the catalase (EC 1.11.1.6) and acid phosphatase (EC 3.1.3.1) activities. Activities of the mitochondrial enzymes NAD+‐isocitrate dehydrogenase (EC 1.1.1.41) and cytochrome c oxidase (EC 1.9.3.1) were also measured to determine the distribution of the endogenous S° between mitochondria and cytoplasm. In young dormant α‐spores, endogenous S° was mostly found in the cytoplasmic lipid reserves, which were mainly phospholipids. The mitochondrial fraction of these young α‐spores contained ca 10% of the total endogenous S°, whereas in aging α‐spores stored for 105 days the endogenous S° was mainly (ca 90%) localized in the mitochondrial fraction. This accumulation of S° in mitochondria of aging α‐spores was correlated with a sharp decrease in phospholipid reserves, endogenous and exogenous respiratory activities, ATP concentration, uptake of sulfate, and NAD+‐isocitrate dehydrogenase and cytochrome c oxidase activities. These metabolic changes were correlated with an irreversible loss of germination capacity which leads to the natural death of P. viticola α‐spores. During the first min of the breaking of dormancy, the young α‐spores possess a 7.3‐fold capacity to reduce exogenous S° with production of hydrogen sulfide, as compared to the aging α‐spores. In young α‐spores the production of hydrogen sulfide was almost totally inhibited by 40 μM antimycin A (92%), and strongly inhibited by 2mM azide (75%) and by 15 μM 2,4‐dinitrophenol (63%). Our work suggests that endogenous S° plays a key role in the regulation of the dormancy and aging processes of α‐spores of P. viticola. [ABSTRACT FROM AUTHOR]

Mechanical work, ATP, ADP, PC, free creatine and lactate concentrations were determined on IAA poisoned frog sartorii tetanically stimulated in humidified N, at 10°C in isotonic conditions (0.25 or 0.45 P). Tetanus duration was 0.35 s, number of tetani was varied from 0 (rest) to 25 (exhaustion). The mechanical work performed per mole ATP+PC split (W) amounted on the average to 16.7 kJ/mol. It was observed, however, that w increased from about 13 to about 24 kJ/mol with decreasing ATP concentration from about 2 (resting value) to about 1 μmol/g and that this decrease in ATP was associated with a decrease of the shortening (and relaxation) speed of the muscle to about 30% of the values observed on the first tetanus. It is concluded that the thermodynamic efficiency of muscle contraction, calculated from the ratio of w (measured) to the thermodynamic affinity (free energy change) of ATP hydrolysis (estimated) increases from about 0.3 to about 0.5 with decreasing ATP concentration and shortening speed. [ABSTRACT FROM AUTHOR]