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It is commonly accepted that dietary nucleotides should be considered as essential nutrients originating mainly but not exclusively from meat and fish dishes. Most research in food science related to nutrition nucleotides is focused on raw products, while the effects of thermal processing of ready-to-eat food on nucleotide content are largely overlooked by the scientific community. The aim of this study is to investigate the impact of thermal processing and cold storage on the content of dietary nucleotides in freshly prepared and canned ready-to-eat meat and fish food. The concentrations of ATP, ADP, AMP, IMP, Ino, and Hx were determined using NMR, HPLC, FPMLC, and ATP bioluminescence analytical techniques; freshness indices K and K1 and adenylate energy charge (AEC) values were estimated to assess the freshness status and confirm a newly unveiled phenomenon of the reappearance of adenylate nucleotides. It was found that in freshly prepared at 65 °C ≤ T ≤ +100 °C and canned food, the concentration of free nucleotides was in the range of 0.001–0.01 µmol/mL and remained unchanged for a long time during cold storage; the correct distribution of mole fractions of adenylates corresponding to 0 < AEC < 0.5 was observed compared to 0.2 < AEC < 1.0 in the original raw samples, with either a high or low content of residual adenylates. It could be assumed that heating at nonenzymatic temperatures T > 65 °C can rupture cell membranes and release residual intracell nucleotides in quite a meaningful concentration. These findings may lead to a conceptual change in the views on food preparation processes, taking into account the phenomenon of the free adenylates renaissance and AEC bioenergetics. [ABSTRACT FROM AUTHOR]

It is commonly accepted that dietary nucleotides should be considered as essential nutrients originating mainly but not exclusively from meat and fish dishes. Most research in food science related to nutrition nucleotides is focused on raw products, while the effects of thermal processing of ready-to-eat food on nucleotide content are largely overlooked by the scientific community. The aim of this study is to investigate the impact of thermal processing and cold storage on the content of dietary nucleotides in freshly prepared and canned ready-to-eat meat and fish food. The concentrations of ATP, ADP, AMP, IMP, Ino, and Hx were determined using NMR, HPLC, FPMLC, and ATP bioluminescence analytical techniques; freshness indices K and K1 and adenylate energy charge (AEC) values were estimated to assess the freshness status and confirm a newly unveiled phenomenon of the reappearance of adenylate nucleotides. It was found that in freshly prepared at 65 °C ≤ T ≤ +100 °C and canned food, the concentration of free nucleotides was in the range of 0.001–0.01 µmol/mL and remained unchanged for a long time during cold storage; the correct distribution of mole fractions of adenylates corresponding to 0 < AEC < 0.5 was observed compared to 0.2 < AEC < 1.0 in the original raw samples, with either a high or low content of residual adenylates. It could be assumed that heating at nonenzymatic temperatures T > 65 °C can rupture cell membranes and release residual intracell nucleotides in quite a meaningful concentration. These findings may lead to a conceptual change in the views on food preparation processes, taking into account the phenomenon of the free adenylates renaissance and AEC bioenergetics.

The hyporheic zone (HZ) is a sensitive ecotone in river ecosystems because of its biodiversity and susceptibility to human activities. Hyporheic fauna are exposed to multiple stressors that affect the physiology and metabolism of organisms and ultimately ecosystem functioning and biodiversity. This study aimed to quantify adenine nucleotides in the stygophilous freshwater amphipod Synurella ambulans and to evaluate the potential of adenylate energy charge (AEC) as a physiological biomarker of general stress in the HZ of the Sava River at sites with different intensities of environmental and anthropogenic stress. Field studies were conducted seasonally (in December 2018 and April, July, and October 2019) at two sampling sites, one upstream (1-UP) and the other downstream (2-DOWN) of the discharge from the wastewater treatment plant using the standard piston pump. The amphipod population from site 1-UP had significantly higher AEC values than the population from site 2-DOWN in all seasons except summer. Coordinated changes in AEC values with the ATP/ADP ratio indicated differences in energy status between the two populations. However, no changes in the apparent equilibrium constant of adenylate kinase were observed, except in spring at site 1-UP. Multiple linear regression models showed the strongest associations of AEC with Fe and Zn accumulated in amphipods, followed by environmental factors (conductivity, dissolved O2, and concentrations of nitrites and phosphates in the interstitial water). AEC was shown to be a useful index of environmental stress in S. ambulans because it can directly measure the change in available energy and thus the metabolic stress to which the organism is exposed. Finally, seasonal and spatial variations in AEC values reflected ecological status in the HZ. [ABSTRACT FROM AUTHOR]

Gaseous factors affect post-harvest physiological processes in horticultural crops, including ornamental flowers. However, the molecular responses of cut flowers to the low-oxygen conditions associated with modified atmosphere packaging (MAP) have not yet been elucidated. Here, we show that storage of cut carnation flowers in a sealed polypropylene bag decreased the oxygen concentration in the bag to 3–5% and slowed flower opening. The vase life of carnation flowers after storage for seven days under MAP conditions was comparable to that without storage and was improved by the application of a commercial-quality preservative. The adenylate energy charge (AEC) was maintained at high levels in petals from florets stored under MAP conditions. This was accompanied by the upregulation of four hypoxia-related genes, among which the HYPOXIA-RESPONSIVE ETHYLENE RESPONSE FACTOR and PHYTOGLOBIN genes (DcERF19 and DcPGB1) were newly identified. These results suggest that hypoxia-responsive genes contribute to the maintenance of the energy status in carnation flowers stored under MAP conditions, making this gas-controlling technique potentially effective for maintaining cut flower quality without cooling. [ABSTRACT FROM AUTHOR]

Precise and accurate quantification is a prerequisite for interpretation of targeted metabolomics data, but this task is challenged by the inherent instability of the analytes. The sampling, quenching, extraction, and sample purification conditions required to recover and stabilize metabolites in representative extracts have also been proven highly dependent on species-specific properties. For Escherichia coli, unspecific leakage has been demonstrated for conventional microbial metabolomics sampling protocols. We herein present a fast filtration-based sampling protocol for this widely applied model organism, focusing on pitfalls such as inefficient filtration, selective loss of biomass, matrix contamination, and membrane permeabilization and leakage. We evaluate the effect of and need for removal of extracellular components and demonstrate how residual salts can challenge analytical accuracy of hyphenated mass spectrometric analyses, even when sophisticated correction strategies are applied. Laborious extraction procedures are bypassed by direct extraction in cold acetonitrile:water:methanol (3:5:2, v/v%), ensuring compatibility with sample concentration and thus, any downstream analysis. By applying this protocol, we achieve and demonstrate high precision and low metabolite turnover, and, followingly, minimal perturbation of the inherent metabolic state. This allows us to herein report absolute intracellular concentrations in E. coli and explore its central carbon metabolome at several commonly applied cultivation conditions. [ABSTRACT FROM AUTHOR]

Background: Prolonging the shelf‐life of liquid‐preserved semen without compromising its fertilizing capacity may increase the efficiency of artificial insemination in pigs. Many fertilization‐relevant processes are adenosine triphosphate dependent. The impact of semen storage and rewarming to body temperature on the energy status of spermatozoa is as yet unknown. Objectives: To investigate the energy status of boar spermatozoa during storage and subsequent rewarming and to reveal the potential role of mitochondrial function for reactivation and maintenance of sperm motility. Materials and methods: Extended semen samples (n = 7 boars) were used. Spermatozoa were challenged by storage at 17°C for 7 days and incubation at 38°C for 180 min. The adenosine triphosphate concentration and energy charge in semen samples and lactate concentration in the extracellular medium were assessed. Viability and mitochondrial activity were determined by flow cytometry, and clustered single‐cell analysis of motility parameters was performed. Results: The energy status was not affected by semen storage (p > 0.05). Rewarming resulted in a net reduction in adenosine triphosphate concentration, which increased with storage time (maximum Day 5: ‐24.2 ± 10.3%) but was not accompanied by a loss in viability, motility, or mitochondrial activity. Blocking glycolysis with 2‐deoxy‐d‐glucose prevented the re‐establishment of motility and mitochondrial activity after rewarming. Mitochondrial activity gradually subsided in virtually all spermatozoa during incubation at 38°C, while adenosine triphosphate and energy charge remained high. Concomitantly, extracellular lactate levels rose, and sperm populations with lower velocity, increased linearity, and low lateral head displacement grew larger. Size changes for major sperm subpopulations correlated with the percentage of viable spermatozoa with high mitochondrial activity (r = 0.44–0.70 for individual subpopulations, p < 0.01). Conclusion: Storage of boar spermatozoa increases the demand of adenosine triphosphate for reactivation of spermatozoa towards fast, non‐linear, and hyperactivation‐like motility patterns upon rewarming. Maintenance of glycolysis seems to be decisive for sperm function after long‐term storage in vitro. [ABSTRACT FROM AUTHOR]

In natural environments, the spectral composition of incident light is often subject to drastic changes due to the abundance of suspended particles, floating animals, and plants in coastal waters. In this study, after four months of culturing under blue light (NB), orange light (NY), dark environment (ND), and natural light (NN), the shell length and weight-specific growth rate in Pacific abalone, Haliotis discus hannai, were ranked in the following order: NY > NN > ND > NB. To understand the growth differences in abalone under these different light environments, we first performed 24-h video monitoring and found that the cumulative movement distance and duration were lowest in group NB, whereas the cumulative movement distance and duration were significantly higher in group ND than in any other group (P NN > ND > NB. According to in situ hybridization analysis, the three subtype genes were widely expressed in the hepatopancreas. Finally, the contents of many lipid metabolites differed significantly among groups and the expression levels of the triglyceride hydrolysis-related gene hormone sensitive lipase and fatty acid oxidation-related gene carnitine palmitoyltransferase 1 were higher in groups ND and NB than in groups NN and NY according to fluorescence quantification PCR (P

The hyporheic zone (HZ) is a sensitive ecotone in river ecosystems because of its biodiversity and susceptibility to human activities. Hyporheic fauna are exposed to multiple stressors that affect the physiology and metabolism of organisms and ultimately ecosystem functioning and biodiversity. This study aimed to quantify adenine nucleotides in the stygophilous freshwater amphipod Synurella ambulans and to evaluate the potential of adenylate energy charge (AEC) as a physiological biomarker of general stress in the HZ of the Sava River at sites with different intensities of environmental and anthropogenic stress. Field studies were conducted seasonally (in December 2018 and April, July, and October 2019) at two sampling sites, one upstream (1-UP) and the other downstream (2-DOWN) of the discharge from the wastewater treatment plant using the standard piston pump. The amphipod population from site 1-UP had significantly higher AEC values than the population from site 2-DOWN in all seasons except summer. Coordinated changes in AEC values with the ATP/ADP ratio indicated differences in energy status between the two populations. However, no changes in the apparent equilibrium constant of adenylate kinase were observed, except in spring at site 1-UP. Multiple linear regression models showed the strongest associations of AEC with Fe and Zn accumulated in amphipods, followed by environmental factors (conductivity, dissolved O2, and concentrations of nitrites and phosphates in the interstitial water). AEC was shown to be a useful index of environmental stress in S. ambulans because it can directly measure the change in available energy and thus the metabolic stress to which the organism is exposed. Finally, seasonal and spatial variations in AEC values reflected ecological status in the HZ.

Gaseous factors affect post-harvest physiological processes in horticultural crops, including ornamental flowers. However, the molecular responses of cut flowers to the low-oxygen conditions associated with modified atmosphere packaging (MAP) have not yet been elucidated. Here, we show that storage of cut carnation flowers in a sealed polypropylene bag decreased the oxygen concentration in the bag to 3–5% and slowed flower opening. The vase life of carnation flowers after storage for seven days under MAP conditions was comparable to that without storage and was improved by the application of a commercial-quality preservative. The adenylate energy charge (AEC) was maintained at high levels in petals from florets stored under MAP conditions. This was accompanied by the upregulation of four hypoxia-related genes, among which the HYPOXIA-RESPONSIVE ETHYLENE RESPONSE FACTOR and PHYTOGLOBIN genes (DcERF19 and DcPGB1) were newly identified. These results suggest that hypoxia-responsive genes contribute to the maintenance of the energy status in carnation flowers stored under MAP conditions, making this gas-controlling technique potentially effective for maintaining cut flower quality without cooling.

The phosphorous (P) is an essential element for several functions, including the synthesis of high energy compounds in cells like adenosine triphosphate (ATP) and arginine phosphate (ArgP). The dietary requirements of P could change according to physiological and environmental conditions, particularly if organisms are exposed to stress conditions. As the P availability could be related to its chemical form in the diet, the present study analyzed the influence of different P sources (KH 2 PO 4 , NaH 2 PO 4 , NH 4 H 2 PO 4 , Na 5 P 3 O 10), on the stress response of the Pacific white shrimp Litopenaeus vannamei. Final weight was 8.5% higher (p < 0.01) in shrimp fed on tripolyphosphate (Na 5 P 3 O 10) diet compared with the monophosphate diets. Weight gain and specific growth also trend to increase, whereas feed conversion ratio trend to decrease with this polyphosphate source, although not significantly. Stress response of shrimp exposed to handling followed by hypoxia was characterized by an overall increase of lactate and glucose levels in hemolymph, as well as a decrease in Adenylate Energy Charge (AEC) and ArgP in the muscle. No effect of dietary P sources was observed on baseline biochemical composition and overall indicators of stress response, except for higher carbohydrate levels in hepatopancreas and RNAm expression of arginine kinase (AK) in muscle with the NaH 2 PO 4 diet, suggesting a potentially better energy status with this diet. • Dietary phosphate source (different salts) does not affect zootechnical performance. • Supplementation of phosphate as sodium salt seems to improve shrimp energy status. • Dietary polyphosphate slightly improved zootechnical performance but did not affect stress response. • Chasing followed by hypoxia represents a good model of induced stress in shrimp. [ABSTRACT FROM AUTHOR]

Adenine phosphoribosyltransferase (APRT) is the key enzyme involved in purine salvage by the incorporation of adenine and phosphoribosyl pyrophosphate to provide adenylate nucleotides. To evaluate the role of APRT in the repair processes of cutaneous wounds in healthy skin and in diabetic patients, a diabetic mouse model (db/db) and age‐matched wild‐type mice were used. Moreover, the topical application of adenine was assessed. In vitro studies, analytical, histological, and immunohistochemical methods were used. Diabetic mice treated with adenine exhibited elevated ATP levels in organismic skin and accelerated wound healing. In vitro studies showed that APRT utilized adenine to rescue cellular ATP levels and proliferation from hydrogen peroxide‐induced oxidative damage. HPLC‐ESI‐MS/MS‐based analysis of total adenylate nucleotides in NIH‐3T3 fibroblasts demonstrated that adenine addition enlarged the cellular adenylate pool, reduced the adenylate energy charge, and provided additional AMP for the further generation of ATP. These data indicate an upregulation of APRT in skin wounds, highlighting its role during the healing of diabetic wounds through regulation of the nucleotide pool after injury. Furthermore, topical adenine supplementation resulted in an enlargement of the adenylate pool needed for the generation of ATP, an important molecule for wound repair. [ABSTRACT FROM AUTHOR]

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) (AK) is the enzyme that catalyzes the reversible conversion of MgATP + AMP to MgADP + ADP, a principal step in adenine nucleotide metabolism and cellular energy homeostasis. To enrich the information about fish AKs, we isolated this enzyme from the lateral muscle of bastard halibut Paralichthys olivaceus and biochemically characterized it. The halibut enzyme (PoAK) extracted from the lateral muscle was purified by column chromatographies on TOYOPEARL SP-650 M and Superdex 75 10/300. The purified PoAK showed a single protein band of ~ 22 kDa on SDS–polyacrylamide gel electrophoresis, and optimal temperature and pH at around 40 °C and 7, respectively. PoAK was appreciably heat stable, e.g., the temperature that caused 50% inactivation during 30-min incubation was 54 °C. The molar ratio for ATP:ADP:AMP in the equilibrium state of the reaction was ~ 1:1:1. Peptide mass fingerprinting indicated that PoAK is the product of adenylate kinase isoform 1 gene (GenBank, XP_019937160.1) encoded in the halibut genome. The deduced amino-acid sequence of the halibut AK comprised 194 residues and showed 92, 91 and 81% amino-acid identities to those of a putative rainbow trout AK, a carp AK and a chicken AK, respectively. [ABSTRACT FROM AUTHOR]

Abstract Background Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are prominent candidate biocatalysts that, together, can enable the direct biotic conversion of lignocellulosic biomass to ethanol. The imbalance and suboptimal turnover rates of redox cofactors are currently hindering engineering efforts to achieve higher bioproductivity in both organisms. Measuring relevant intracellular cofactor concentrations will help understand redox state of these cofactors and help identify a strategy to overcome these limitations; however, metabolomic determinations of these labile metabolites have historically proved challenging. Results Through our validations, we verified the handling and storage stability of these metabolites, and verified extraction matrices and extraction solvent were not suppressing mass spectrometry signals. We recovered adenylate energy charge ratios (a main quality indicator) above 0.82 for all extractions. NADH/NAD+ values of 0.26 and 0.04 for an adhE-deficient strain of C. thermocellum and its parent, respectively, reflect the expected shift to a more reduced redox potential when a species lacks the ability to re-oxidize NADH by synthesizing ethanol. This method failed to yield reliable results with C. bescii and poor-growing strains of T. saccharolyticum. Conclusions Our validated protocols demonstrate and validate the extraction and analysis of selected redox and energy-related metabolites from two candidate consolidated bioprocessing biocatalysts, C. thermocellum and T. saccharolyticum. This development and validation highlights the important, but often neglected, need to optimize and validate metabolomic protocols when adapting them to new cell or tissue types.

Adenine nucleotides—adenosine monophosphate, diphosphate, and triphosphate—are of utmost importance to all living organisms, where they play a critical role in the energy metabolism and are tied to allosteric regulation in various regulatory enzymes. Adenylate energy charge represents the precise relationship between the concentrations of adenosine monophosphate, diphosphate, and triphosphate and indicates the amount of metabolic energy available to an organism. The experimental conditions of adenylate extraction in freshwater amphipod Gammarus fossarum are reported here for the first time and are crucial for the qualitative and quantitative determination of adenylate nucleotides using efficient and sensitive ion-pair reverse phase LC. It was shown that amphipod calcified exoskeleton impeded the neutralization of homogenate. The highest adenylate yield was obtained by homogenization in perchloric acid and subsequent addition of potassium hydroxide and phosphate buffer to achieve a pH around 11. This method enables separation and accurate detection of adenylates. Our study provides new insight into the complexity of adenylate extraction and quantification that is crucial for the application of adenylate energy charge as a confident physiological measure of environmental stress and as a health index of G. fossarum.

Free magnesium (Mg2+) is a signal of the adenylate (ATP+ADP+AMP) status in the cells. It results from the equilibrium of adenylate kinase (AK), which uses Mg-chelated and Mg-free adenylates as substrates in both directions of its reaction. The AK-mediated primary control of intracellular [Mg2+] is finely interwoven with the operation of membrane-bound adenylate- and Mg2+-translocators, which in a given compartment control the supply of free adenylates and Mg2+ for the AK-mediated equilibration. As a result, [Mg2+] itself varies both between and within the compartments, depending on their energetic status and environmental clues. Other key nucleotide-utilizing/producing enzymes (e.g., nucleoside diphosphate kinase) may also be involved in fine-tuning of the intracellular [Mg2+]. Changes in [Mg2+] regulate activities of myriads of Mg-utilizing/requiring enzymes, affecting metabolism under both normal and stress conditions, and impacting photosynthetic performance, respiration, phloem loading and other processes. In compartments controlled by AK equilibrium (cytosol, chloroplasts, mitochondria, nucleus), the intracellular [Mg2+] can be calculated from total adenylate contents, based on the dependence of the apparent equilibrium constant of AK on [Mg2+]. Magnesium signaling, reflecting cellular adenylate status, is likely widespread in all eukaryotic and prokaryotic organisms, due simply to the omnipresent nature of AK and to its involvement in adenylate equilibration.

Many important marine species are suffering declines. Release of pollutants into aquatic environments are likely contributors to this decline, and so biochemical methods to study the effects of sublethal toxicant exposure in marine organisms are urgently needed. The goal of this dissertation has been to develop non-invasive 31P-nuclear magnetic resonance (NMR) techniques to evaluate the effects of toxicant exposure on energy metabolism in marine invertebrates. Red abalone (Haliotis rufescens), the basis for an important commercial and sport fishery were chosen to serve as a model for other invertebrates due to the general physiological adaptations, such as their ability to survive extended periods of hypoxia, possessed by these animals, as well as their sedentary nature and large homogeneous foot muscle, which makes them ideal organisms for in vivo NMR spectroscopy.Initial studies examined changes in adenosine triphosphate (ATP), phosphoarginine (PA), inorganic phosphate, intracellular pH, and intracellular free Mg2+ in response to exposure to the mitochondrial stressors hypoxia, pentachlorophenol (PCP), and sodium azide. These studies were also adapted for use with a larval abalone stage, and the effects of PCP exposure was examined in these animals.Further studies refined these techniques to examine the rate of ATP formation from PA via the arginine kinase reaction using a magnetization transfer approach. Stress application increased rates of ATP formation, in contrast to mammalian systems, where declines in ATP formation are generally noted.In the final study the sensitivity of the classical biochemical parameter adenylate energy charge (AEC) was compared with NMR derived measurements of cytosolic adenosine diphosphate (ADP) in response to the three stressors. Cytosolic ADP increased two-three fold in response to stress application while AEC showed little or no change. The effect of increasing ADP on arginine kinase flux is also discussed.The work provided in this dissertation demonstrates some of the utility of in vivo approaches in evaluating the effects of sublethal stress exposure, and suggests future environmental applications in identifying currently unrecognized populations of marine organisms subject to toxicant induced stress.

Background: Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are prominent candidate biocatalysts that, together, can enable the direct biotic conversion of lignocellulosic biomass to ethanol. The imbalance and suboptimal turnover rates of redox cofactors are currently hindering engineering efforts to achieve higher bioproductivity in both organisms. Measuring relevant intracellular cofactor concentrations will help understand redox state of these cofactors and help identify a strategy to overcome these limitations; however, metabolomic determinations of these labile metabolites have historically proved challenging. Results: Through our validations, we verified the handling and storage stability of these metabolites, and verified extraction matrices and extraction solvent were not suppressing mass spectrometry signals. We recovered adenylate energy charge ratios (a main quality indicator) above 0.82 for all extractions. NADH/NAD+ values of 0.26 and 0.04 for an adhE-deficient strain of C. thermocellum and its parent, respectively, reflect the expected shift to a more reduced redox potential when a species lacks the ability to re-oxidize NADH by synthesizing ethanol. This method failed to yield reliable results with C. bescii and poor-growing strains of T. saccharolyticum. Conclusions: Our validated protocols demonstrate and validate the extraction and analysis of selected redox and energy-related metabolites from two candidate consolidated bioprocessing biocatalysts, C. thermocellum and T. saccharolyticum. This development and validation highlights the important, but often neglected, need to optimize and validate metabolomic protocols when adapting them to new cell or tissue types. [ABSTRACT FROM AUTHOR]

The presence of a mixture of progestogens at ng/L concentration levels in surface waters is a worldwide problem. Only a few studies explore the effect of progestogen treatment in a mixture as opposed to individual chemicals to shed light on how non-target species respond to these contaminants. In the present study, we used an invertebrate model species, Lymnaea stagnalis , exposed to a mixture of four progestogens (progesterone, levonorgestrel, drospirenone, and gestodene) in 10 ng/L concentration for 3 weeks. Data at both physiological and cellular/molecular level were analyzed using the ELISA technique, stereomicroscopy combined with time lapse software, and capillary microsampling combined with mass spectrometry. The treatment of adult Lymnaeas caused reduced egg production, and low quality egg mass on the first week, compared to the control. Starting from the second week, the egg production, and the quality of egg mass were similar in both groups. At the end of the third week, the egg production and the vitellogenin-like protein content of the hepatopancreas were significantly elevated in the treated group. At the cellular level, accelerated cell proliferation was observed during early embryogenesis in the treated group. The investigation of metabolomic changes resulted significantly elevated hexose utilization in the single-cell zygote cytoplasm, and elevated adenylate energy charge in the egg albumen. These changes suggested that treated snails provided more hexose in the eggs in order to improve offspring viability. Our study contributes to the knowledge of physiological effect of equi-concentration progestogen mixture at environmentally relevant dose on non-target aquatic species. [ABSTRACT FROM AUTHOR]

Recently, a novel culture system consisting of primary hepatocytes structured over a network of endothelial cells on the Engelbreth-Holm-Swarm (EHS) gel has been reported. This in vitro liver model on the EHS gel (IVLEHS) has been shown to maintain the expression of hepatic genes and their functional activity. Moreover, the IVLEHS was more sensitive to xenobiotics than hepatocyte monocultures, suggesting the potential utility of this culture system for compound hepatotoxicity screening. However, the effect of this three-dimensional structure formation on the cellular metabolic profile of hepatocytes in the IVLEHS is not well understood. To address this concern, we performed metabolome analysis using capillary electrophoresis-time of flight mass spectrometry. Between the IVLEHS and mono-cultured hepatocytes on the EHS gel, there was no significant difference in the levels of metabolites of the urea cycle and the tricarboxylic acid cycle, essential amino acids, and adenylate energy charge (AEC) which is an important indicator of cellular energy status. On the other hand, acetaminophen-dependent decrease of the AEC in the IVLEHS was greater than that in the monoculture, suggesting the higher sensitivity of IVLEHS to acetaminophen-induced hepatotoxicity which is caused by metabolic activation of this drug. Further analysis showed that the levels of taurocholate, one of the major conjugated bile acids, were higher in the IVLEHS than in the monoculture. Considering that the construction of the IVLEHS did not seem to disturb the major cellular metabolism, our findings would strengthen the concept that IVLEHS would have beneficial effects on the maintenance of hepatic functions. [ABSTRACT FROM AUTHOR]

The reformed European Common Fisheries Policy introduced a discard ban, with a possibility of exempting species where a high discard survival can be demonstrated. This necessitates a validation of the methods used for estimating the discard mortality of candidate species. In this study, we assess whether reflex impairment can predict short-term mortality in commercially trawled European plaice upon landing and after air exposure of up to 90min. Sub-lethal stress was assessed by a suite of physiological variables. Over a 10-day period, mortality was monitored for a total of 199 plaice following trawl and air exposure of varying duration, and for 50 control fish scored for reflex impairment on board the vessel. Mortality was only observed in fish exposed to air for >60 min, and averaged 11.1% (95% CI = 7.1-16.3%). Reflex impairment was found to be a significant (P < 0.001) predictor of mortality in a generalized linear model, excluding other initially included variables by using a stepwise method. Plasma cortisol, haematocrit, and plasma osmolality all indicated a profound and increasing level of stress with air exposure, accompanied by a near depletion of muscle phosphocreatine and nucleotides. Fishing site had an unexpected, but significant (p < 0.05) effect on stress levels, which was also reflected in reflex impairment and mortality. Based on these findings, a possible exemption from the discard ban should include considerations on the duration of air exposure. [ABSTRACT FROM AUTHOR]

Current technologies allowing the use of extremely low oxygen partial pressures ( p O 2 ), the introduction of 1-methylcyclopronene (1-MCP), and the regulatory prohibition of diphenylamine are changing the conventional storage protocols for pear cultivars. Internal disorders, in particular, severely damage pear quality during controlled atmosphere storage. ‘Rocha’ pear ( Pyrus cummunis L.) was stored for 136 d at −0.5 °C in air or under 3.0 and 0.5 kPa O 2 with 0.6 kPa CO 2 . Fruits treated with 150 nL L −1 1-MCP were also stored at 3.0 and 0.5 kPa O 2 after 32 d in air following the treatment. Internal disorders did not develop in fruit stored in air (20.8 kPa O 2 ) or at 0.5 kPa O 2 but affected 10.2% of the fruit stored in 3.0 kPa O 2 after 136 d. 1-MCP increased disorder incidence at 0.5 and at 3.0 kPa O 2 . Four types of internal disorders occurred: core browning, white cavity, necrotic cavity, and flesh browning. Low O 2 reduced ethylene production and respiration rates which were further reduced by the treatment with 1-MCP. ATP concentration and adenylate energy charge were higher in fruit stored in air than in those at 3.0 and were generally lowest in fruit at 0.5 kPa O 2 . The effect of p O 2 on energy metabolism prevailed over that of 1-MCP treatment. The linkage between ATP and adenylate energy charge (AEC) and the incidence of internal disorders was not strong, since under the same p O 2 , 1-MCP enhanced the incidence of disorders with a negligible effect on adenylate nucleotides or AEC. It was not possible to establish a threshold of ATP concentration or AEC below which internal disorder develop. In conclusion, poststorage quality of ‘Rocha’ pear was better at the extremely low p O 2 of 0.5 kPa than at 3.0 kPa. 1-MCP was detrimental to internal disorders and blocked poststorage softening of ‘Rocha’ pear stored at 0.5 kPa O 2 . [ABSTRACT FROM AUTHOR]

Pear ( Pyrus communis L.) is a climacteric fruit whose ripening behavior is highly cultivar-dependent. This study investigates the postharvest ripening physiology and biochemistry of ‘Rocha’ pear treated with 1-methylcyclopropene (1-MCP). Fruit from a single orchard harvested at the mature-green stage were treated with 0 (control), 150, and 300 nL L −1 1-MCP, and allowed to ripen in air at 20 °C. ‘Rocha’ pear without exposure to chilling ripened with a typical pattern of ethylene production and respiration rates. Inhibition of ethylene action by 1-MCP delayed the ethylene production peak and reduced its intensity by 60%. ATP concentration in control fruit was maximum after 7 d and declined toward the end of the ripening period. 1-MCP induced a transient reduction of ATP concentrations and adenylate energy charge (AEC) during ripening. AEC of control fruit increased slightly during the first week and decreased to a stable value of 0.7 toward the end of ripening. Skin color and firmness of control fruit changed faster during the first week of ripening. 1-MCP induced a delaying in softening, in yellowing and in electrolyte efflux changes, but not impaired the ripening progress. In conclusion, ‘Rocha’ pear ripened immediately after harvest without chilling treatment or exogenous ethylene application and adjusted the AEC during ripening even with lower respiration rates induced by 1-MCP. [ABSTRACT FROM AUTHOR]