Pseudochrobactrum asaccharolyticum mitigates arsenic induced oxidative stress of maize plant by enhancing water status and antioxidant defense system.

BACKGROUND: Oxidative stress mediated by reactive oxygen species (ROS) is a common denominator in arsenic toxicity. Arsenic stress in soil affects the water absorption, decrease stomatal conductance, reduction in osmotic, and leaf water potential, which restrict water uptake and osmotic stress in plants. Arsenic-induced osmotic stress triggers the overproduction of ROS, which causes a number of germination, physiological, biochemical, and antioxidant alterations. Antioxidants with potential to reduce ROS levels ameliorate the arsenic-induced lesions. Plant growth promoting rhizobacteria (PGPR) increase the total soluble sugars and proline, which scavenging OH radicals thereby prevent the oxidative damages cause by ROS. The main objective of this study was to evaluate the potential role of Arsenic resistant PGPR in growth of maize by mitigating arsenic stress. METHODOLOGY: Arsenic tolerant PGPR strain MD3 (Pseudochrobactrum asaccharolyticum) was used to dismiss the 'As' induced oxidative stress in maize grown at concentrations of 50 and 100 mg/kg. Previously isolated arsenic tolerant bacterial strain MD3 "Pseudochrobactrum asaccharolyticum was used for this experiment. Further, growth promoting potential of MD3 was done by germination and physio-biochemical analysis of maize seeds. Experimental units were arranged in Completely Randomized Design (CRD). A total of 6 sets of treatments viz., control, arsenic treated (50 & 100 mg/kg), bacterial inoculated (MD3), and arsenic stress plus bacterial inoculated with three replicates were used for Petri plates and pot experiments. After treating with this MD3 strain, seeds of corn were grown in pots filled with or without 50 mg/kg and 100 mg/kg sodium arsenate. RESULTS: The plants under arsenic stress (100 mg/kg) decreased the osmotic potential (0.8 MPa) as compared to control indicated the osmotic stress, which caused the reduction in growth, physiological parameters, proline accumulation, alteration in antioxidant enzymes (Superoxide dismutase-SOD, catalase-CAT, peroxidase-POD), increased MDA content, and H2O2 in maize plants. As-tolerant Pseudochrobactrum asaccharolyticum improved the plant growth by reducing the oxidation stress and antioxidant enzymes by proline accumulation. PCA analysis revealed that all six treatments scattered differently across the PC1 and PC2, having 85.51% and 9.72% data variance, respectively. This indicating the efficiency of As-tolerant strains. The heatmap supported the As-tolerant strains were positively correlated with growth parameters and physiological activities of the maize plants. CONCLUSION: This study concluded that Pseudochrobactrum asaccharolyticum reduced the 'As' toxicity in maize plant through the augmentation of the antioxidant defense system. Thus, MD3 (Pseudochrobactrum asaccharolyticum) strain can be considered as bio-fertilizer.

AlgU mediates hyperosmotic tolerance in Pseudomonas protegens SN15-2 by regulating membrane stability, ROS scavenging, and osmolyte synthesis.

Pseudomonas protegens can serve as an agricultural biocontrol agent. P. protegens often encounters hyperosmotic stress during industrial production and field application. The ability of P. protegens to withstand hyperosmotic stress is important for its application as a biocontrol agent. AlgU is a global regulator responsible for stress response and biocontrol ability. However, the specific regulatory role of AlgU in the hyperosmotic adaptation of P. protegens is poorly understood. In this study, we found that the AlgU mutation disrupted the hyperosmotic tolerance of P. protegens. Many genes and metabolites related to cell envelope formation were significantly downregulated in ΔalgU compared with that in the wild-type (WT) strain under hyperosmotic conditions, and we found that the algU mutation caused membrane integrity to be compromised and increased membrane permeability. Further experiments revealed that the cell envelope integrity protein TolA, which is regulated by AlgU, contributes to cell membrane stability and osmotic tolerance in P. protegens. In addition, several genes related to oxidative stress response were significantly downregulated in ΔalgU, and higher levels of intracellular reactive oxygen species were found in ΔalgU. Furthermore, we found that the synthesis of N-acetyl glutaminyl glutamine amide is directly regulated by AlgU and contributes to the hyperosmotic adaptation of P. protegens. This study revealed the mechanisms of AlgU's participation in osmotic tolerance in P. protegens, and it provides potential molecular targets for research on the hyperosmotic adaptation of P. protegens.IMPORTANCEIn this study, we found that the extracytoplasmic function sigma factor AlgU is essential for the survival of P. protegens under hyperosmotic conditions. We provided evidence supporting the roles of AlgU in influencing cell membrane stability, intracellular reactive oxygen species (ROS) accumulation, and dipeptide N-acetylglutaminylglutamine amide (NAGGN) synthesis in P. protegens under hyperosmotic conditions. Our findings revealed the mechanisms of AlgU's participation in hyperosmotic stress tolerance in P. protegens, and they provide potential molecular targets for research on the hyperosmotic adaptation of P. protegens, which is of value in improving the biocontrol ability of P. protegens.

The Roles of the Anthraquinone Parietin in the Tolerance to Desiccation of the Lichen Xanthoria parietina: Physiology and Anatomy of the Pale and Bright-Orange Thalli.

Lichens are symbiotic organisms that effectively survive in harsh environments, including arid regions. Maintaining viability with an almost complete loss of water and the rapid restoration of metabolism during rehydration distinguishes lichens from most eukaryotic organisms. The lichen Xanthoria parietina is known to have high stress tolerance, possessing diverse defense mechanisms, including the presence of the bright-orange pigment parietin. While several studies have demonstrated the photoprotective and antioxidant properties of this anthraquinone, the role of parietin in the tolerance of lichens to desiccation is not clear yet. Thalli, which are exposed to solar radiation and become bright orange, may require enhanced desiccation tolerance. Here, we showed differences in the anatomy of naturally pale and bright-orange thalli of X. parietina and visualized parietin crystals on the surface of the upper cortex. Parietin was extracted from bright-orange thalli by acetone rinsing and quantified using HPLC. Although acetone rinsing did not affect PSII activity, thalli without parietin had higher levels of lipid peroxidation and a lower membrane stability index in response to desiccation. Furthermore, highly pigmented thalli possess thicker cell walls and, according to thermogravimetric analysis, higher water-holding capacities than pale thalli. Thus, parietin may play a role in desiccation tolerance by stabilizing mycobiont membranes, providing an antioxidative defense, and changing the morphology of the upper cortex of X. parietina.

StICE1 enhances plant cold tolerance by directly upregulating StLTI6A expression.

KEY MESSAGE: Under cold conditions, StICE1 enhances plant cold tolerance by upregulating StLTI6A expression to maintain the cell membrane stability. Cold stress affects potato plants growth and development, crop productivity and quality. The ICE-CBF-COR regulatory cascade is the well-known pathway in response to cold stress in plants. ICE1, as a MYC-like bHLH transcription factor, can regulate the expressions of CBFs. However, whether ICE1 could regulate other genes still need to be explored. Our results showed that overexpressing ICE1 from potato in Arabidopsis thaliana could enhance plant cold tolerance. Under cold stress, overexpressed StICE1 in plants improved the stability of cell membrane, enhanced scavenging capacity of reactive oxygen species and increased expression levels of CBFs and COR genes. Furthermore, StICE1 could bind to the promoter of StLTI6A gene, which could maintain the stability of the cell membrane, to upregulate StLTI6A expression under cold conditions. Our findings revealed that StICE1 could directly regulate StLTI6A, CBF and COR genes expression to response to cold stress.

Donkey semen cryopreservation: Alternatives with permeable, non-permeable cryoprotectants and seminal plasma.

Cryopreservation of semen is an important technique to preserve genetic material. Yet, pregnancy rates in jennies after artificial insemination with frozen-thawed donkey semen are poor. This condition has been attributed to the impact of permeable cryoprotectants, that could cause high post-breeding endometritis. Removal of seminal plasma (SP) prior to semen freezing process is another contributing factor. SP is involved in a multitude of sperm functions and events preceding fertilization and has a mediating effect of sperm capacitation and postcoital uterine inflammatory response. The aim of this study was to evaluate different alternatives in donkey semen cryopreservation with permeable, non-permeable cryoprotectants, BSA and SP. Thirty ejaculates from 10 donkeys were cryopreserved with different combinations of dimethylformamide (DMF, 5%), sucrose (SUC, 200 mM) and homologous SP (10%): DMF (T1), DMF/SP (T2), SUC/BSA (T3), SUC/BSA/SP (T4), DMF/SUC/BSA (T5), DMF/SUC/BSA/SP (T6), DMF/BSA (T7) and DMF/BSA/SP (T8). After thawing, sperm motility and kinetics were assessed by computerized semen analysis. Sperm vitality (SV) was evaluated by fluorescence microscopy, functional membrane integrity (FMI) by the HOST test, abnormal morphology by eosin-nigrosin staining and sperm membrane stability by flow cytometry. For statistical analysis, sperm quality indexes (SQi) were obtained, general linear models were carried out and mean comparisons were made by the Tukey test. T1, T2, T5, T6, and T7 had higher and equivalent results for motility, most kinetic parameters and function membrane integrity. Cryopreservation of donkey semen without permeable cryoprotectant (T3 and T4) showed a reduction in motility, kinetics, SV, FMI and SQi. T5 showed a reduction in progressive motility, sperm velocities, IMF and SQi compared to other DMF treatments. T6 and T8 achieved higher SQi values compared to T1, but they were not different compared to T2 and T7. T1 had a smaller sperm population with low-M540 compared to T3. It is concluded that the use of permeable cryoprotectant is essential to achieve higher post-thaw quality of donkey semen. In addition, the combined use of BSA, SUC and/or PS may provide additional sperm protection compared to the individual use of DMF.

Proteomic analysis of spermatozoa reveals caseins play a pivotal role in preventing short-term periods of subfertility in stallions†.

Stallions experience transient fluctuations in fertility throughout the breeding season. Considering pregnancy diagnoses cannot be ascertained until ~14 days postbreeding, the timely detection of decreases in stallion fertility would enhance industry economic and welfare outcomes. Therefore, this study aimed to identify the proteomic signatures reflective of short-term fertility fluctuations and to determine the biological mechanisms governing such differences. Using liquid chromatography-mass spectrometry (LC-MS/MS), we compared the proteomic profile of semen samples collected from commercially "fertile" stallions, during high- and low-fertility periods. A total of 1702 proteins were identified, of which, 38 showed a significant change in abundance (P ≤ 0.05). Assessment of intra- and interstallion variability revealed that caseins (namely κ-, α-S1-, and α-S2-casein) were significantly more abundant during "high-fertility" periods, while several epididymal, and seminal plasma proteins (chiefly, epididymal sperm binding protein 1 [ELSPbP1], horse seminal plasma protein 1 [HSP-1], and clusterin), were significantly more abundant during "low-fertility" periods. We hypothesized that an increased abundance of caseins offers greater protection from potentially harmful seminal plasma proteins, thereby preserving cell functionality and fertility. In vitro exposure of spermatozoa to casein resulted in decreased levels of lipid scrambling (Merocyanine 540), higher abundance of sperm-bound caseins (α-S1-, α-S2-, and κ-casein), and lower abundance of sperm-bound HSP-1 (P ≤ 0.05). This study demonstrates key pathways governing short-term fertility fluctuations in the stallion, thereby providing a platform to develop robust, fertility assessment strategies into the future.

Trehalose: a promising osmo-protectant against salinity stress-physiological and molecular mechanisms and future prospective.

Salt stress is one of the leading threats to crop growth and productivity across the globe. Salt stress induces serious alterations in plant physiological, metabolic, biochemical functioning and it also disturbs antioxidant activities, cellular membranes, photosynthetic performance, nutrient uptake and plant water uptake and resulting in a significant reduction in growth and production. The application of osmoprotectants is considered as an important strategy to induce salt tolerance in plants. Trehalose (Tre) has emerged an excellent osmolyte to induce salinity tolerance and it got considerable attention in recent times. Under salinity stress, Tre helps to maintain the membrane integrity, and improves plant water relations, nutrient uptake and reduces the electrolyte leakage and lipid per-oxidation. Tre also improves gas exchange characteristics, protects the photosynthetic apparatus from salinity induced oxidative damages and brings ultra-structure changes in the plant body to induce salinity tolerance. Moreover, Tre also improves antioxidant activities and expression of stress responsive proteins and genes and confers salt tolerance in plants. Additionally, Tre is also involved in signaling association with signaling molecules and phytohormones and resultantly improved the plant performance under salt stress. Thus, it is interesting to understand the role of Tre in mediating the salinity tolerance in plants. Therefore, in this review we have summarized the different physiological and molecular roles of Tre to induce salt tolerance in plants. Moreover, we have also provided the information on Tre cross-talk with various osmolytes and hormones, and its role in stress responsive genes and antioxidant activities. Lastly, we also shed light on research gaps that need to be addressed in future studies. Therefore, this review will help the scientists to learn more about the Tre in changing climate conditions and it will also provide new insights to insights that could be used to develop salinity tolerance in plants.

Recovery of Hydrochloric Acid from Industrial Wastewater by Diffusion Dialysis Using a Spiral-Wound Module.

In the present study, the possibility of using a spiral-wound diffusion dialysis module was studied for the separation of hydrochloric acid and Zn2+, Ni2+, Cr3+, and Fe2+ salts. Diffusion dialysis recovered 68% of free HCl from the spent pickling solution contaminated with heavy-metal-ion salts. A higher volumetric flowrate of the stripping medium recovered a more significant portion of free acid, namely, 77%. Transition metals (Fe, Ni, Cr) apart from Zn were rejected by >85%. Low retention of Zn (35%) relates to the diffusion of negatively charged chloro complexes through the anion-exchange membrane. The mechanical and transport properties of dialysis FAD-PET membrane under accelerated degradation conditions was investigated. Long-term tests coupled with the economic study have verified that diffusion dialysis is a suitable method for the treatment of spent acids, the salts of which are well soluble in water. Calculations predict significant annual OPEX savings, approximately up to 58%, favouring diffusion dialysis for implementation into wastewater management.

Salicylic Acid Stimulates Defense Systems in Allium hirtifolium Grown under Water Deficit Stress.

Nowadays, the use of the growth regulator salicylic acid for improving a plant's resistance to environmental stresses such as drought is increasing. The present study investigated the effect of salicylic acid on the physiological traits, antioxidant enzymes, yield, and quality of Allium hirtifolium (shallots) under drought conditions for three years (2016-2017, 2017-2018, and 2018-2019). The experiment was conducted as a split-plot based on a randomized complete block design with four repeats. Irrigation as the main factor in four levels of 100% (full irrigation), 75% and 50% of the plant water requirements with non-irrigation (dryland), and salicylic acid as the sub-factor in four levels of 0, 0.75, and 1 mmol, were the studied factors in this research. The combined analysis of three-year data showed that drought reduced leaf relative water content (RWC), membrane stability index (MSI), chlorophyll content, onion yield, and increased activity of antioxidant enzymes, proline content, tang, and allicin of shallots. Shallot spraying with salicylic acid improved leaf RWC, MSI, chlorophyll content, and onion yield. The highest yield of onion (1427 gr m-2) belonged to full irrigation and foliar application of 1 mmol salicylic acid. The lowest yield (419.8 gr m-2) belonged to plats with non-irrigation and non-application of salicylic acid. By improving the effective physiological traits in resistance to water deficit, salicylic acid adjusted the effects of water deficit on the yield of shallots. Foliar application of 1 mmol salicylic acid in dryland and irrigation of 50% of the plant water requirement increased onion yield by 15.12% and 29.39%, respectively, compared to the control treatment without salicylic acid.

Exogenous melatonin mitigates chromium toxicity in maize seedlings by modulating antioxidant system and suppresses chromium uptake and oxidative stress.

Melatonin, being an endogenous signaling molecule plays important role in plant growth and stress alleviation. The present study was conducted to evaluate the ameliorative role of melatonin against Cr toxicity in maize seedlings. The Cr toxicity (50, 100 and 200 µM) severely affected hydroponically grown seedlings growth in a dose-dependent manner; however, the melatonin (0.5 and 1.0 µM) application markedly restored toxicity-induced growth retardation. Higher dose of melatonin (1.0 µM) was more effective in case of lower Cr toxicity (50 and 100 µM). Exposure of 200 µM Cr caused 45% and 43% reduction in shoot and root lengths and more than 80% reduction in biomass. In case of 200 µM Cr toxicity, application of 1.0 µM MT effectively restored shoot and root lengths reduction (from 45 to 30%) and biomass decline (from 80 to around 60%). Biomass restoration by 1.0 µM melatonin under 50 and 100 µM Cr was even more pronounced bringing it near to control plants having no Cr exposure. Further, both melatonin levels also improved root tips, root diameter, root volume and root surface area that had been damaged by Cr exposure. The melatonin also alleviated Cr-induced chlorophyll and carotenoids inhibition, improved relative water content, and markedly lowered proline and MDA content in shoots. Lower accumulation of MDA and proline, and greater membrane stability indices indicate that the melatonin conferred better plant growth by playing the role of antioxidant and detoxifying oxidative stress creating substances. Although antioxidant enzymes viz. SOD, POD, CAT and APX activities were also elevated by MT, this increase was not significantly different in the most of cases. No significant difference in NPK contents of shoot was observed by Cr and melatonin application indicating the growth retardation being caused directly by Cr intrinsic toxicity and not by nutrients deficiency. The melatonin-based amelioration of Cr toxicity in maize seedlings seems as the result of its nature as antioxidant, and not by activation/elevation of antioxidative enzymatic system.

Temperature adaptations of the thermophilic snail Echinolittorina malaccana: insights from metabolomic analysis.

The periwinkle snail Echinolittorina malaccana, for which the upper lethal temperature is near 55°C, is one of the most heat-tolerant eukaryotes known. We conducted a multi-level investigation - including cardiac physiology, enzyme activity, and targeted and untargeted metabolomic analyses - that elucidated a spectrum of adaptations to extreme heat in this organism. All systems examined showed heat intensity-dependent responses. Under moderate heat stress (37-45°C), the snail depressed cardiac activity and entered a state of metabolic depression. The global metabolomic and enzymatic analyses revealed production of metabolites characteristic of oxygen-independent pathways of ATP generation (lactate and succinate) in the depressed metabolic state, which suggests that anaerobic metabolism was the main energy supply pathway under heat stress (37-52°C). The metabolomic analyses also revealed alterations in glycerophospholipid metabolism under extreme heat stress (52°C), which likely reflected adaptive changes to maintain membrane structure. Small-molecular-mass organic osmolytes (glycine betaine, choline and carnitine) showed complex changes in concentration that were consistent with a role of these protein-stabilizing solutes in protection of the proteome under heat stress. This thermophilic species can thus deploy a wide array of adaptive strategies to acclimatize to extremely high temperatures.

6-Benzylaminopurine Alleviates the Impact of Cu2+ Toxicity on Photosynthetic Performance of Ricinus communis L. Seedlings.

Copper (Cu) is an essential element involved in various metabolic processes in plants, but at concentrations above the threshold level, it becomes a potential stress factor. The effects of two different cytokinins, kinetin (KIN) and 6-benzylaminopurine (BAP), on chlorophyll a fluorescence parameters, stomatal responses and antioxidation mechanisms in castor (Ricinus communis L.) under Cu2+ toxicity was investigated. Ricinus communis plants were exposed to 80 and 160 µM CuSO4 added to the growth medium. Foliar spraying of 15 µM KIN and BAP was carried out on these seedlings. The application of these cytokinins enhanced the tissue water status, chlorophyll contents, stomatal opening and photosynthetic efficiency in the castor plants subjected to Cu2+ stress. The fluorescence parameters, such as Fm, Fv/Fo, Sm, photochemical and non-photochemical quantum yields, energy absorbed, energy trapped and electron transport per cross-sections, were more efficiently modulated by BAP application than KIN under Cu2+ toxicity. There was also effective alleviation of reactive oxygen species by enzymatic and non-enzymatic antioxidation systems, reducing the membrane lipid peroxidation, which brought about a relative enhancement in the membrane stability index. Of the various treatments, 80 µM CuSO4 + BAP recorded the highest increase in photosynthetic efficiency compared to other cytokinin treatments. Therefore, it can be concluded that BAP could effectively alleviate the detrimental effects of Cu2+toxicity in cotyledonary leaves of R. communis by effectively modulating stomatal responses and antioxidation mechanisms, thereby enhancing the photosynthetic apparatus' functioning.

Certain, but Not All, Tetraether Lipids from the Thermoacidophilic Archaeon Sulfolobus acidocaldarius Can Form Black Lipid Membranes with Remarkable Stability and Exhibiting Mthk Channel Activity with Unusually High Ca2+ Sensitivity.

Bipolar tetraether lipids (BTL) have been long thought to play a critical role in allowing thermoacidophiles to thrive under extreme conditions. In the present study, we demonstrated that not all BTLs from the thermoacidophilic archaeon Sulfolobus acidocaldarius exhibit the same membrane behaviors. We found that free-standing planar membranes (i.e., black lipid membranes, BLM) made of the polar lipid fraction E (PLFE) isolated from S. acidocaldarius formed over a pinhole on a cellulose acetate partition in a dual-chamber Teflon device exhibited remarkable stability showing a virtually constant capacitance (~28 pF) for at least 11 days. PLFE contains exclusively tetraethers. The dominating hydrophobic core of PLFE lipids is glycerol dialky calditol tetraether (GDNT, ~90%), whereas glycerol dialkyl glycerol tetraether (GDGT) is a minor component (~10%). In sharp contrast, BLM made of BTL extracted from microvesicles (Sa-MVs) released from the same cells exhibited a capacitance between 36 and 39 pF lasting for only 8 h before membrane dielectric breakdown. Lipids in Sa-MVs are also exclusively tetraethers; however, the dominating lipid species in Sa-MVs is GDGT (>99%), not GDNT. The remarkable stability of BLMPLFE can be attributed to strong PLFE-PLFE and PLFE-substrate interactions. In addition, we compare voltage-dependent channel activity of calcium-gated potassium channels (MthK) in BLMPLFE to values recorded in BLMSa-MV. MthK is an ion channel isolated from a methanogenic that has been extensively characterized in diester lipid membranes and has been used as a model for calcium-gated potassium channels. We found that MthK can insert into BLMPLFE and exhibit channel activity, but not in BLMSa-MV. Additionally, the opening/closing of the MthK in BLMPLFE is detectable at calcium concentrations as low as 0.1 mM; conversely, in diester lipid membranes at such a low calcium concentration, no MthK channel activity is detectable. The differential effect of membrane stability and MthK channel activity between BLMPLFE and BLMSa-MV may be attributed to their lipid structural differences and thus their abilities to interact with the substrate and membrane protein. Since Sa-MVs that bud off from the plasma membrane are exclusively tetraether lipids but do not contain the main tetraether lipid component GDNT of the plasma membrane, domain segregation must occur in S. acidocaldarius. The implication of this study is that lipid domain formation is existent and functionally essential in all kinds of cells, but domain formation may be even more prevalent and pronounced in hyperthermophiles, as strong domain formation with distinct membrane behaviors is necessary to counteract randomization due to high growth temperatures while BTL in general make archaea cell membranes stable in high temperature and low pH environments whereas different BTL domains play different functional roles.

CK19 stabilizes CFTR at the cell surface by limiting its endocytic pathway degradation.

Protein interactions that stabilize the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) at the apical membranes of epithelial cells have not yet been fully elucidated. We identified keratin 19 (CK19 or K19) as a novel CFTR-interacting protein. CK19 overexpression stabilized both wild-type (WT)-CFTR and Lumacaftor (VX-809)-rescued F508del-CFTR (where F508del is the deletion of the phenylalanine residue at position 508) at the plasma membrane (PM), promoting Cl- secretion across human bronchial epithelial (HBE) cells. CK19 prevention of Rab7A-mediated lysosomal degradation was a key mechanism in apical CFTR stabilization. Unexpectedly, CK19 expression was decreased by ∼40% in primary HBE cells from homogenous F508del patients with CF relative to non-CF controls. CK19 also positively regulated multidrug resistance-associated protein 4 expression at the PM, suggesting that this keratin may regulate the apical expression of other ATP-binding cassette proteins as well as CFTR.-Hou, X., Wu, Q., Rajagopalan, C., Zhang, C., Bouhamdan, M., Wei, H., Chen, X., Zaman, K., Li, C., Sun, X., Chen, S., Frizzell, R. A., Sun, F. CK19 stabilizes CFTR at the cell surface by limiting its endocytic pathway degradation.

Benzotriazole alleviates copper mediated lysosomal membrane damage and antioxidant defense system responses in earthworms (Eisenia fetida).

Benzotriazole (BTR) is a common corrosion inhibitor used to protect copper (Cu) and Cu alloys. To reveal the combined subacute toxicity of BTR and Cu at environmental levels on terrestrial animals, the activity of antioxidative enzymes and the glutathione levels in earthworms (Eisenia fetida) of the single or co-exposure treatments were determined. The activity of both antioxidant enzymes and non-enzymatic antioxidants was affected by BTR in earthworms. Moreover, the analyses of lysosomal neutral red retention time and total antioxidant capacity indicated a detoxification effect of BTR on Cu-induced impairments of the antioxidant defense capacity in earthworms. The apoptotic rate of coelomocytes in earthworms of the co-exposure treatment was lower than that in earthworms treated with Cu only, indicating that BTR alleviates Cu mediated lysosomal membrane damage and antioxidant defense system responses in earthworms.

Neighbour signals perceived by phytochrome B increase thermotolerance in Arabidopsis.

Due to the preeminence of reductionist approaches, understanding of plant responses to combined stresses is limited. We speculated that light-quality signals of neighbouring vegetation might increase susceptibility to heat shocks because shade reduces tissue temperature and hence the likeness of heat shocks. In contrast, plants of Arabidopsis thaliana grown under low-red/far-red ratios typical of shade were less damaged by heat stress than plants grown under simulated sunlight. Neighbour signals reduce the activity of phytochrome B (phyB), increasing the abundance of PHYTOCHROME-INTERACTING FACTORS (PIFs). The phyB mutant showed high tolerance to heat stress even under simulated sunlight, and a pif multiple mutant showed low tolerance under simulated shade. phyB and red/far-red ratio had no effects on seedlings acclimated with nonstressful warm temperatures before the heat shock. The phyB mutant showed reduced expression of several fatty acid desaturase (FAD) genes and less proportion of fully unsaturated fatty acids and electrolyte leakage of membranes exposed to heat shocks. Red-light-activated phyB also reduced thermotolerance of dark-grown seedlings but not via changes in FADs expression and membrane stability. We propose that the reduced photosynthetic capacity linked to thermotolerant membranes would be less costly under shade, where the light input limits photosynthesis.

Regulation and function of the flavonoid-inducible efflux system, emrR-emrAB, in Agrobacterium tumefaciens C58.

The expression of the Agrobacterium tumefaciens emrAB operon, which encodes a membrane fusion protein and an inner membrane protein, is inducible by various flavonoids, including apigenin, genistein, luteolin, naringenin, and quercetin. Among these flavonoids, quercetin is the best inducer, followed by genistein. The emrR gene is divergently transcribed from the emrAB operon. The EmrR protein, which belongs to the TetR transcriptional regulator family, negatively regulates the expression of emrAB and of itself. Electrophoretic mobility shift assays and DNase I footprinting showed that EmrR binds directly at two EmrR-binding sites in the emrR-emrAB intergenic region and that quercetin inhibits the DNA-binding activity of EmrR. Promoter-lacZ fusion analyses and 5' rapid amplification of cDNA ends were performed to map the emrR and emrAB promoters. Compared with the wild-type strain, the emrA mutant strain exhibited similar levels of resistance to the tested antibiotics. In contrast, disruption of emrR conferred protection against nalidixic acid and novobiocin, but it rendered A. tumefaciens sensitive to tetracycline and erythromycin. The emrR mutation also destabilized the outer membrane of A. tumefaciens, resulting in increased sensitivity to SDS and low pH. These findings demonstrate that proper regulation of emrR-emrAB is required for free-living A. tumefaciens to survive in deleterious environments in which toxic compounds are present. Nonetheless, A. tumefaciens strains that lack emrR or emrA still have the ability to cause tumors when infecting Nicotiana benthamiana plants.

Effect of ingesting carbohydrate only or carbohydrate plus casein protein hydrolysate during a multiday cycling race on left ventricular function, plasma volume expansion and cardiac biomarkers.

PURPOSE: Multiday racing causes mild left ventricular (LV) dysfunction from day 1 that persists on successive days. We evaluated ingesting casein protein hydrolysate-carbohydrate (PRO) compared with carbohydrate-only (CHO) during a 3-day mountain bike race. METHODS: Eighteen male cyclists were randomly assigned to ingest 6.7% carbohydrate without (CHO) or with 1.3% casein hydrolysate (PRO) during racing (~ 4-5 h/day; 68/71/71 km). Conventional LV echocardiography, plasma albumin content, plasma volume (PV) and blood biomarkers were measured before day 1 and post race on day 3. RESULTS: Fourteen cyclists (n = 7 per group) completed the race. PV increased in CHO (mean increase (95% CI), 10.2% (0.1 to 20.2)%, p = 0.045) but not in PRO (0.4% (- 6.1 to 6.9)%). Early diastolic transmitral blood flow (E) was unchanged but deceleration time from peak E increased post race (CHO: 46.7 (11.8 to 81.6) ms, p = 0.019; PRO: 24.2 (- 0.5 to 48.9) ms, p = 0.054), suggesting impaired LV relaxation. Tissue Doppler mitral annular velocity was unchanged in CHO, but in PRO septal early-to-late diastolic ratio decreased (p = 0.016) and was compensated by increased lateral early (p = 0.034) and late (p = 0.012) velocities. Systolic function was preserved in both groups; with increased systolic lateral wall velocity in PRO (p = 0.002). Effect size increase in serum creatine kinase (CK) activity, CK-MB and C-reactive protein concentrations was less in PRO than CHO (Cohen's d mean ± SD, PRO: 2.91 ± 2.07; CHO: 7.56 ± 4.81, p = 0.046). CONCLUSION: Ingesting casein hydrolysate with carbohydrate during a 3-day race prevented secondary hypervolemia and failed to curb impaired LV relaxation despite reducing tissue damage and inflammatory biomarkers. Without PV expansion, systolic function was preserved by lateral wall compensating for septal wall dysfunction.

Effect of the Incorporation of Functionalized Cyclodextrins in the Liposomal Bilayer.

Liposomes loaded with drug­cyclodextrin complexes are widely used as drug delivery systems, especially for species with low aqueous solubility and stability. Investigation of the intimate interactions of macrocycles with liposomes are essential for formulation of efficient and stable drug-in-cyclodextrin-in-liposome carriers. In this work, we reported the preparation of unilamellar vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) embedded with native ß-cyclodextrin and two synthetic derivatives: heptakis(2,3,6-tri-O-methyl)-ß-cyclodextrin (TMCD) and heptakis(2,3-di-O-acetyl)-ß-cyclodextrin (DACD). We then studied the effect of these macrocycles on the liposomal size, membrane viscosity, and liposomal stability at different temperatures and concentrations. We observed that TMCD and DACD affected vesicle size and the change of size was related to CD concentration. Irrespective of its nature, the macrocycle established interactions with the phospholipidic head groups, preventing cyclodextrins to diffuse into the lipid bilayer, as confirmed by molecular dynamics simulations. Such supramolecular structuring improves liposome stability making these colloid systems promising carriers for biologically active compounds.

Effect of ubiquinone-10 on the stability of biomimetic membranes of relevance for the inner mitochondrial membrane.

Ubiquinone-10 (Q10) plays a pivotal role as electron-carrier in the mitochondrial respiratory chain, and is also well known for its powerful antioxidant properties. Recent findings suggest moreover that Q10 could have an important membrane stabilizing function. In line with this, we showed in a previous study that Q10 decreases the permeability to carboxyfluorescein (CF) and increases the mechanical strength of 1-palmitoyl-2-oleyl-sn-glycero-phosphocholine (POPC) membranes. In the current study we report on the effects exerted by Q10 in membranes having a more complex lipid composition designed to mimic that of the inner mitochondrial membrane (IMM). Results from DPH fluorescence anisotropy and permeability measurements, as well as investigations probing the interaction of liposomes with silica surfaces, corroborate a membrane stabilizing effect of Q10 also in the IMM-mimicking membranes. Comparative investigations examining the effect of Q10 and the polyisoprenoid alcohol solanesol on the IMM model and on membranes composed of individual IMM components suggest, moreover, that Q10 improves the membrane barrier properties via different mechanisms depending on the lipid composition of the membrane. Thus, whereas Q10's inhibitory effect on CF release from pure POPC membranes appears to be directly and solely related to Q10's lipid ordering and condensing effect, a mechanism linked to Q10's ability to amplify intrinsic curvature elastic stress dominates in case of membranes containing high proportions of palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE).