Tracking W-Formate Dehydrogenase Structural Changes During Catalysis and Enzyme Reoxidation

Metal-dependent formate dehydrogenases (Fdh) catalyze the reversible conversion of CO₂ to formate, with unrivalled efficiency and selectivity. However, the key catalytic aspects of these enzymes remain unknown, preventing us from fully benefiting from their capabilities in terms of biotechnological applications. Here, we report a time-resolved characterization by X-ray crystallography of the Desulfovibrio vulgaris Hildenborough SeCys/W-Fdh during formate oxidation. The results allowed us to model five different intermediate structures and to chronologically map the changes occurring during enzyme reduction. Formate molecules were assigned for the first time to populate the catalytic pocket of a Fdh. Finally, the redox reversibility of DvFdhAB in crystals was confirmed by reduction and reoxidation structural studies.     

Relevance of the Exocyst in Arabidopsis exo70e2 Mutant for Cellular Homeostasis under Stress

Plants must adapt to cope with adverse environmental conditions that affect their growth and development. To overcome these constraints, they can alter their developmental patterns by modulating cellular processes and activating stress-responsive signals. Alongside the activation of the antioxidant (AOX) system, a high number of genes are expressed, and proteins must be distributed to the correct locations within the cell. The endomembrane system and associated vesicles thus play an important role. Several pathways have been associated with adverse environmental conditions, which is the case for the exocyst-positive organelle—EXPO. The present work, using Arabidopsis mutants with T-DNA insertions in the gene EXO70, essential for EXPO vesicles formation, was designed to characterise the anatomical (morphology and root length), biochemical (quantification of stress markers and antioxidant system components), and molecular responses (gene expression) to abiotic stresses (saline, drought, oxidative, and metal-induced toxicity). The results obtained showed that mutant plants behave differently from the wild type (WT) plants. Therefore, in the exo70 mutant, morphological changes were more noticeable in plants under stress, and the non-enzymatic component of the antioxidant system was activated, with no alterations to the enzymatic component. Furthermore, other defence strategies, such as autophagy, did not show important changes. These results confirmed the EXPO as an important structure for tolerance/adaptation to stress.     

Dipotassium Glycyrrhizinate on Melanoma Cell Line: Inhibition of Cerebral Metastases Formation by Targeting NF-kB Genes-Mediating MicroRNA-4443 and MicroRNA-3620—Dipotassium Glycyrrhizinate Effect on Melanoma

Glycyrrhizic acid (GA), a natural compound isolated from licorice (Glycyrrhiza glabra), has exhibited anti-inflammatory and anti-tumor effects in vitro. Dipotassium glycyrrhizinate (DPG), a dipotassium salt of GA, also has shown an anti-tumor effect on glioblastoma cell lines, U87MG and T98G. The study investigated the DPG effects in the melanoma cell line (SK-MEL-28). MTT assay demonstrated that the viability of the cells was significantly decreased in a time- and dose-dependent manner after DPG (IC₅₀ = 36 mM; 24 h). DNA fragmentation suggested that DPG (IC₅₀) induced cellular apoptosis, which was confirmed by a significant number of TUNEL-positive cells (p-value = 0.048) and by PARP-1 [0.55 vs. 1.02 arbitrary units (AUs), p-value = 0.001], BAX (1.91 vs. 1.05 AUs, p-value = 0.09), and BCL-2 (0.51 vs. 1.07 AUs, p-value = 0.0018) mRNA compared to control cells. The proliferation and wound-healing assays showed an anti-proliferative effect on DPG-IC₅₀-treated cells, also indicating an inhibitory effect on cell migration (p-values < 0.001). Moreover, it was observed that DPG promoted a 100% reduction in melanospheres formation (p-value = 0.008). Our previous microRNAs (miRs) global analysis has revealed that DPG might increase miR-4443 and miR-3620 expression levels. Thus, qPCR showed that after DPG treatment, SK-MEL-28 cells presented significantly high miR-4443 (1.77 vs. 1.04 AUs, p-value = 0.02) and miR-3620 (2.30 vs. 1.00 AUs, p-value = 0.01) expression compared to control cells, which are predicted to target the NF-kB, CD209 and TNC genes, respectively. Both genes are responsible for cell attachment and migration, and qPCR revealed significantly decreased CD209 (1.01 vs. 0.54 AUs, p-value = 0.018) and TNC (1.00 vs. 0.31 AUs, p-value = 2.38 × 10⁻⁶) mRNA expression levels after DPG compared to untreated cells. Furthermore, the migration of SK-MEL-28 cells stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) was attenuated by adding DPG by wound-healing assay (48 h: p-value = 0.004; 72 h: p-value = 7.0 × 10⁻⁴). In addition, the MMP-9 expression level was inhibited by DPG in melanoma cells stimulated by TPA and compared to TPA-treated cells (3.56 vs. 0.99 AUs, p-value = 0.0016) after 24 h of treatment. Our results suggested that DPG has an apoptotic, anti-proliferative, and anti-migratory effect on SK-MEL-28 cells. DPG was also able to inhibit cancer stem-like cells that may cause cerebral tumor formation.     

A comparative study of the purification of betanin

Betanin is a natural pigment with antioxidant properties used as a food colourant. This work describes the spectrophotometric and chromatographic quantification of betanin (2S/15S) and its epimer isobetanin (2S/15R) in fresh beetroot juice, food-grade beetroot powder and betanin standard diluted in dextrin. Absorption spectra of all three samples were deconvoluted using a mixed three-function model. Food-grade beetroot powder has the largest amount of violet-red impurities, probably formed during processing. The purification of betanin from these complex matrices was carried out by seven different methods. Ion exchange chromatography was the most efficient method for the purification of betanin from all samples; however, fractions contain high amounts of salt. Reversed-phase HPLC as well as reversed-phase column chromatography also produced good results at a much faster rate. The longer retention time of isobetanin when compared to betanin in reversed-phase conditions has been investigated by means of quantum-mechanical methods.     

A Rapid and Eco-Friendly Method for Determination of Hydrolysable Tannins and Its Application to Honey Samples

This paper describes the development of a rapid and clean method for the determination of hydrolysable tannins in honey samples, focusing on an easy and green sample pretreatment and low generation of waste. The proposed method is based on the reaction between KIO₃ and hydrolysable tannin compounds in acetate buffer medium (pH 4.75), with formation of a colored product measured at 525 nm. The analytical conditions were optimized using experimental design (central composite design). The linear range obtained was 40.0–740 mg L^?1 (r?=?0.998), and the detection (LOD) and quantification (LOQ) limits were 12.2 and 40.8 mg L^?1, respectively. Acetate buffer was used to ensure formation of the colored product obtained by reaction between the chromogenic reagent and the analyte. The method showed good results when applied to honey samples, with recoveries in the range of 78.3–96.1 %, and the use of diffuse reflectance spectroscopy coupled with spot test analysis provided low reagent consumption and minimized waste generation. The results obtained with the proposed method were confirmed by a spectrophotometric method.     

Effect of RF magnetron sputtering parameters on the optimization of the discharge capacity of ternary lithium oxide thin films

Journal of Materials Science: Materials in Electronics - The increasing demand for lithium-ion batteries has stimulated the investigation of new compounds in order to reduce the costs and the...     

Corn grain-processing method interacts with calcium salts of palm fatty acids supplementation on milk production and energy balance of early-lactation cows grazing tropical pasture

The objective of our study was to investigate the associative effects of feeding Ca salts of palm fatty acids (FA) and corn grain-processing method on production, nutrient digestibility, energy balance, and carryover effects of early-lactation dairy cows grazing a tropical pasture. Treatment diets were offered from 3 to 16 wk postpartum (treatment period), in which all cows grazed elephant grass (Pennisetum purpureum L. Cameroon) and treatments were added to a concentrate supplement. Treatments were flint corn grain-processing method either as fine ground (FGC) or steam-flaked (SFC) associated with Ca salts of palm FA supplementation either not supplemented or supplemented (CSPO). From 17 to 40 wk postpartum (carryover period) all cows received a common diet fed as total mixed ration. During the treatment period, a tendency for an interaction between CSPO and corn grain-processing method were observed for milk yield, milk fat yield, and energy-corrected milk (ECM), as CSPO caused them to increased to a greater extent in the FGC diet compared with the SFC diet. Furthermore, a tendency for an interaction between CSPO and corn grain-processing method was observed for body weight change, because CSPO increased body weight loss in the FGC diet but not in the SFC diet. The CSPO increased milk yield, milk fat yield, 3.5% fat-corrected milk, ECM, and cumulative milk yield compared with not supplemented. Also, CSPO increased energy intake, milk energy output, and energy partitioning toward milk, whereas reduced energy was allocated to body reserves. The SFC increased milk yield, ECM, milk protein yield, milk casein yield, and cumulative milk yield, and decreased milk urea N compared with FGC. The SFC compared with FGC also increased body condition score and body weight change, and increased energy partitioning toward body reserves. During the carryover period, an interaction between CSPO and corn grain-processing method was observed for milk yield, which occurred because CSPO maintained higher milk yield in the FGC diet but not in the SFC diet. Therefore, in the carryover period, the additive effect between SFC and CSPO that occurred in the treatment period was not maintained throughout the carryover period. However, CSPO increased yields of milk fat, protein, casein as well as fat-corrected milk and ECM. In conclusion, corn grain-processing method interacts with CSPO supplementation on production responses and carryover effects of grazing cows. When CSPO was fed in the FGC diet, milk production increased to a greater extent than when fed in the SFC diet, but also caused greater mobilization of reserves at early lactation. This suggests an interaction between fat supplementation and corn grain-processing method on energy partitioning of dairy cows. Also, both supplementation with CSPO and SFC were effective strategies to increase energy intake and yields of milk and milk solids. The carryover effect on milk production was greater for CSPO supplementation than corn grain-processing method, whereas feeding SFC diets had lower mobilization of reserves and less body weight and body condition score variation throughout lactation.     

Heuristic approaches for the optimal wiring in large scale robotic skin design

This paper faces the problem of optimizing the wiring and the connections in a tactile skin for robots. The robotic skin is a device composed of a network of tactile sensors, whose wiring can be very complex: the control of this complexity is a key problem. In the considered robotic skin, skin elements are grouped into skin patches, which output tactile data that have to be read by a micro-controller. The logical connections between the sensors must be defined in order to route signals through the network. A finite set of micro-controllers is given and a set of constraints is imposed on the given assignment and routing. The considered problem has a combinatorial nature and it can be formulated as a Minimum Constrained Spanning Forest problem with costs on arcs that cannot be a priori defined as they are solution-dependent. The problem is NP-hard. The paper introduces a mathematical formulation and then proposes a Multi-Start Heuristic algorithm and an Ant Colony Optimization approach whose effectiveness is evaluated through experimental tests performed on both real and synthetically generated instances.