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.     

Prediction of Drug Synergism between Peptides and Antineoplastic Drugs Paclitaxel, 5-Fluorouracil,and Doxorubicin Using In Silico Approaches

Chemotherapy is the main treatment for most early-stage cancers; nevertheless, its efficacy is usually limited by drug resistance, toxicity, and tumor heterogeneity. Cell-penetrating peptides (CPPs) are small peptide sequences that can be used to increase the delivery rate of chemotherapeutic drugs to the tumor site, therefore contributing to overcoming these problems and enhancing the efficacy of chemotherapy. The drug combination is another promising strategy to overcome the aforementioned problems since the combined drugs can synergize through interconnected biological processes and target different pathways simultaneously. Here, we hypothesized that different peptides (P1–P4) could be used to enhance the delivery of chemotherapeutic agents into three different cancer cells (HT-29, MCF-7, and PC-3). In silico studies were performed to simulate the pharmacokinetic (PK) parameters of each peptide and antineoplastic agent to help predict synergistic interactions in vitro. These simulations predicted peptides P2–P4 to have higher bioavailability and lower T_max, as well as the chemotherapeutic agent 5-fluorouracil (5-FU) to have enhanced permeability properties over other antineoplastic agents, with P3 having prominent accumulation in the colon. In vitro studies were then performed to evaluate the combination of each peptide with the chemotherapeutic agents as well as to assess the nature of drug interactions through the quantification of the Combination Index (CI). Our findings in MCF-7 and PC-3 cancer cells demonstrated that the combination of these peptides with paclitaxel (PTX) and doxorubicin (DOXO), respectively, is not advantageous over a single treatment with the chemotherapeutic agent. In the case of HT-29 colorectal cancer cells, the combination of P2–P4 with 5-FU resulted in synergistic cytotoxic effects, as predicted by the in silico simulations. Taken together, these findings demonstrate that these CPP6-conjugates can be used as adjuvant agents to increase the delivery of 5-FU into HT-29 colorectal cancer cells. Moreover, these results support the use of in silico approaches for the prediction of the interaction between drugs in combination therapy for cancer.     

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.     

The effect of cocoa fermentation and weak organic acids on growth and ochratoxin A production by Aspergillus species

The acidic characteristics of cocoa beans have influence on flavor development in chocolate. Cocoa cotyledons are not naturally acidic, the acidity comes from organic acids produced by the fermentative microorganisms which grow during the processing of cocoa. Different concentrations of these metabolites can be produced according to the fermentation practices adopted in the farms, which could affect the growth and ochratoxin A production by fungi. This work presents two independent experiments carried out to investigate the effect of some fermentation practices on ochratoxin A production by Aspergillus carbonarius in cocoa, and the effect of weak organic acids such as acetic, lactic and citric at different pH values on growth and ochratoxin A production by A. carbonarius and Aspergillus niger in culture media. A statistical difference (ρ<0.05) in the ochratoxin A level in the cured cocoa beans was observed in some fermentation practices adopted. The laboratorial studies demonstrate the influence of organic acids on fungal growth and ochratoxin A production, with differences according to the media pH and the organic acid present. Acetic acid was the most inhibitory acid against A. carbonarius and A. niger. From the point of view of food safety, considering the amount of ochratoxin A produced, fermentation practices should be conducted towards the enhancement of acetic acid, although lactic and citric acids also have an important role in lowering the pH to improve the toxicity of acetic acid.     

Anti-inflammatory effect of aqueous extracts of roasted and green Coffea arabica L.

Green coffee contains a large quantity and variety of polyphenols and flavonoids. The roasting affects the composition of the polyphenols in coffee, due to the formation of compounds generated by Maillard reaction, which can have anti-inflammatory or antioxidant potential. The anti-inflammatory and antioxidant effects of aqueous extracts of green (AGCa) and roasted (ARCa) coffee beans were investigated in animal models and using a DPPH radical scavenging test. In the formalin test the extracts reduced licking activity only in the late phase. The inhibitory values of oedema at 3 h post-carrageenan were 53% and 74% for 100 and 300 mg/kg of the AGCa extract and 36% for ARCa (300 mg/kg). Leukocyte recruitment into the peritoneal cavity was inhibited by the extracts. The antioxidant activities of the extracts were higher than the reference antioxidants, ascorbic acid and butylated hydroxytoluene. These results indicate that coffee extracts exhibit anti-inflammatory and antioxidant properties.     

Isolation and characterisation of pectic substances from murta (Ugni molinae Turcz) fruits

Cell walls polysaccharides from murta fruit (Ugni molinae Turcz), an endemic Chilean species with relevant food uses, were fractionated by water, ammonium oxalate, hot diluted HCl and cold diluted NaOH extractions. The polysaccharide fractions were analysed for monosaccharide composition and physicochemical properties. Pectic substances were found in all extracts, but mainly in the oxalate and acid soluble fractions, in which they appear as homogalacturonan polymers. Murta pectin was further extracted by hot diluted acid treatment using industrial conditions, yielding 30% by weight of dry fruit. The polymer showed similar composition and physicochemical properties to those of commercial citrus pectin, presenting a galacturonic acid content of 70.9% (w/w), a molecular weight of 597 kDa, and a methoxylation degree of 57%. The FT-IR spectrum of murta pectin suggests the presence of ferulic acid residues on its structure and the NMR analysis confirmed the structure of this polysaccharide. It is concluded that murta fruit can be considered as a valuable source of high quality pectin.     

High-cell-density fermentation of Saccharomyces cerevisiae for the optimisation of mead production

Mead is a traditional drink that contains 8%–18% (v/v) of ethanol, resulting from the alcoholic fermentation of diluted honey by yeasts. Mead fermentation is a time-consuming process and the quality of the final product is highly variable. Therefore, the present investigation had two main objectives: first, to determine the adequate inoculum size of two commercial wine-making strains of Saccharomyces cerevisiae for the optimisation of mead fermentation; and second, to determine if an increase in yeast pitching rates in batch fermentations altered the resulting aroma profiles. Minor differences were detected in the growth kinetics between the two strains at the lowest pitching rate. With increasing pitching rates net growth of the strain ICV D47 progressively decreased, whereas for the QA23 the increasing inoculum size had no influence on its net growth. The time required to reach the same stage of fermentation ranged from 24 to 96 h depending on the inoculum size. The final aroma composition was dependent on the yeast strain and inoculum size. Fourteen of the twenty-seven volatile compounds quantified could contribute to mead aroma and flavour because their concentrations rose above their respective thresholds. The formation of these compounds was particularly pronounced at low pitching rates, except in mead fermented by strain ICV D47, at 10⁶ CFUs/mL. The esters isoamyl acetate, ethyl octanoate and ethyl hexanoate were the major powerful odourants found in the meads. The results obtained in this study demonstrate that yeast strain and inoculum size can favourably impact mead's flavour and aroma profiles.     

Analytical Tools Used to Distinguish Chemical Profiles of Plants Widely Consumed as Infusions and Dietary Supplements: Artichoke,Milk Thistle,and Borututu

Artichoke, borututu, and milk thistle are three medicinal plants widely consumed as infusions or included in dietary supplements (e.g., pills and syrups). Despite the high consumption of these plants, studies on their nutritional value and primary metabolites are scarce, with their composition in secondary metabolites such as phenolic compounds being only reported. Therefore, their nutritional value was assessed, and analytical tools (liquid and gas chromatography coupled to different detectors) were used to distinguish their chemical profiles according to their hydrophilic (sugars and organic acids) and lipophilic (fatty acids and tocopherols) components. Chromatographic techniques are important analytical tools used in the identification and quantification of several molecules, also being a standard requirement to distinguish different profiles. Borututu gave the highest energetic value with the highest content of carbohydrates and fat, sucrose and total sugars, shikimic and citric acids, and α-, β-, δ-, and total tocopherols. Artichoke had the highest ash and protein contents, oxalic acid, SFA (mainly palmitic acid), and γ-tocopherol, and also the best n-6/n-3 ratio. Milk thistle showed the highest levels of fructose and glucose, quinic acid and total organic acids, and PUFA, mainly linoleic acid, and the best PUFA/SFA ratio. The hydrophilic compounds identified in the studied plants, mostly sugars, are responsible for the energy contribution of their widely consumed infusions. Otherwise, the bioactivity of lipophilic compounds, namely, unsaturated fatty acids and tocopherols, is lost in those preparations but can be recovered in dietary supplements based on the plants. As far as we know, this is the first report on the detailed composition of the three plants regarding their nutritional features.     

Effects of ozonized water and heat treatment on the papaya fruit epidermis

This study examined the effects of O₃ and hot water treatments on the epidermis of Golden papaya fruit. Heat treatments were applied in a hot water brushing (HWB) system. Papayas were brushed under a pressurized hot water rinse stage at 45, 55 and 65 °C for 60 s. In the HWB treatment, 4 ppm ozone was applied to the papayas for 1 or 2 min. The results show that ozone applications did not affect the fruit's cuticular surface, while heat treatments allowed natural fissures on the fruit epidermis to recover. Several crystalloid forms were identified on the epidermis of the papayas after the heat treatments. The predominant crystalloid forms on papayas are tubular and there is a positive response to temperature; the higher the temperature, the larger and more frequent the tubular crystalloids.     

Indoor air quality in Portuguese schools: levels and sources of pollutants

Indoor air quality (IAQ) parameters in 73 primary classrooms in Porto were examined for the purpose of assessing levels of volatile organic compounds (VOCs), aldehydes, particulate matter, ventilation rates and bioaerosols within and between schools, and potential sources. Levels of VOCs, aldehydes, PM_2.5, PM₁₀, bacteria and fungi, carbon dioxide (CO₂), carbon monoxide, temperature and relative humidity were measured indoors and outdoors and a walkthrough survey was performed concurrently. Ventilation rates were derived from CO₂ and occupancy data. Concentrations of CO₂ exceeding 1000 ppm were often encountered, indicating poor ventilation. Most VOCs had low concentrations (median of individual species <5 μg/m³) and were below the respective WHO guidelines. Concentrations of particulate matter and culturable bacteria were frequently higher than guidelines/reference values. The variability of VOCs, aldehydes, bioaerosol concentrations, and CO₂ levels between schools exceeded the variability within schools. These findings indicate that IAQ problems may persist in classrooms where pollutant sources exist and classrooms are poorly ventilated; source control strategies (related to building location, occupant behavior, maintenance/cleaning activities) are deemed to be the most reliable for the prevention of adverse health consequences in children in schools.