Validation of a mutant of the pore-forming toxin sticholysin-I for the construction of proteinase-activated immunotoxins

The use of pore-forming toxins from sea anemones (actinoporins) in the construction of immunotoxins (ITs) against tumour cells is an alternative for cancer therapy. However, the main disadvantage of actinoporin-based ITs obtained so far has been the poor cellular specificity associated with the toxin's ability to bind and exert its activity in almost any cell membrane. Our final goal is the construction of tumour proteinase-activated ITs using a cysteine mutant at the membrane binding region of sticholysin-I (StI), a cytolysin isolated from the sea anemone Stichodactyla helianthus. The mutant and the ligand moiety would be linked by proteinase-sensitive peptides through the StI cysteine residue blocking the toxin binding region and hence the IT non-specific killing activity. To accomplish this objective the first step was to obtain the mutant StI W111C, and to evaluate the impact of mutating tryptophan 111 by cysteine on the toxin pore-forming capacity. After proteolysis of the cleavage sequence, a short peptide would remain attached to the toxin. The next step was to evaluate whether this mutant is able to form pores even with a residual peptide linked to cysteine 111. In this work we demonstrated that (i) StI W111C shows pore-forming capacity in a nanomolar range, although it is 8-fold less active than the wild-type recombinant StI, corroborating the previously reported importance of residue 111 for the binding of StI to membranes, and (ii) the mutant is able to form pores even with a residual seven-residue peptide linked to cysteine 111. In addition, it was demonstrated that binding of a large molecule to cysteine 111 renders an inactive toxin that is no longer able to bind to the membrane. These results validate the mutant StI W111C for its use in the construction of tumour proteinase-activated ITs.     

Partial removal of protein associated with arabinoxylans: Impact on the viscoelasticity,crosslinking content,and microstructure of the gels formed

Arabinoxylans (AX) treated with protease and dialyzed (AXP) or only dialyzed (AXD) formed gels showing an increase in the elastic modulus G′ (1291 and 1419 Pa, respectively) and the ferulic acid dimers (3.34 and 3.10 μg/mg polysaccharide, respectively) and trimers (0.51 and 0.53 μg/mg polysaccharide, respectively) in comparison to AX gels (767 Pa, 0.56 and 0.12 μg/mg polysaccharide, respectively). Nevertheless, the G′ values and crosslinking contents were not different among the AXP and AXD gels, suggesting that the amount of protein removed (54%) does not affect these parameters. Confocal laser scanning microscopy analysis showed that AXP treatment promotes the homogeneity of the gels. In addition, scanning electron microscopy observations indicated that AXD and particularly AXP gels had a more compact microstructure. Thus, the partial removal of protein associated with AX does not impact the viscoelasticity and crosslinking content of the gels formed but could improve their microstructural characteristics. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47300.     

Nutritional Value of Various Ray Fish Liver Oils to the Pacific White Shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

A 32-day comparative feeding trial was performed to evaluate the nutritional value of four different ray fish liver oils to the Pacific white shrimp Litopenaeus vannamei. Four feeds were prepared with liver oil extracted from Dasyatis brevis, Rhinoptera steindachneri, Aetobatus narinari, and R. bonasus. A control feed was prepared with Menhaden fish oil. Ray fish liver oils were mainly composed of poly- and highly unsaturated fatty acids and contained levels of 20:5n-3 and 22:6n-3 comparable in magnitude to those of Menhaden fish oil, except for A. narinari liver oil, which had moderately low concentrations of 20:5n-3 and 22:6n-3 but showed a particularly high level of 20:4n-6, more than six times greater than that of Menhaden fish oil. Dietary fatty acids significantly influenced the fatty acid composition of shrimp muscle tissue, e.g., the diet with Menhaden fish oil elicited significantly higher shrimp muscle DHA level than diets with oil from D. brevis, and A. narinari, but not than diets with oil from R. steindachneri and R. bonasus. In spite of these differences, all four ray fish liver oils evaluated were as efficient in promoting growth and survival of L. vannamei as Menhaden fish oil, an ingredient known for its adequate nutritional quality to shrimp and fish. This study demonstrated one of the many possible applications of a locally-available resource that is currently being wasted.     

Ozone Application for Postharvest Disinfection of Tomatoes

Ozone is an effective alternative for the postharvest treatment of fruits and vegetables. The purpose of this study was to evaluate the ozone application in gaseous or aqueous phases for postharvest disinfection of tomatoes (Lycopersicon esculentum Mill) cultivar FA-180. Fruits harvested at the breaker stage were exposed to ozone concentrations of 25 and 45 mg m^?3 for 2 h per day during 16 days, at non-controlled temperature and relative humidity. Exposure to ozone during storage extends the shelf-life of tomatoes, besides preserving its sensory attributes. Mature tomatoes, inoculated with Escherichia coli ATCC 25922, were washed with ozonated water containing 0.5 to 1.0 mg L^?1 during 15 to 30 min. To achieve an adequate disinfection, 1 mg L^?1 and 15 min are recommended. The disinfection with ozonated water was effective.     

Edible films from mucilage of Cereus hildmannianus fruits: Development and characterization

Mucilages are heteropolysaccharides with rich monosaccharide composition. They are capable of forming films that are brittle and fragile, and in order to obtain flexible materials, plasticizers must be added. The present study aims to evaluate the physicochemical properties of mucilage‐based films produced with Cereus hildmannianus and different glycerol concentrations by the casting technique. Transparent and yellowish films were obtained by the addition of glycerol, which also improved handleability, evidenced by increased elongation, and reduced tensile strength and Young's modulus. Thermal gravimetric analysis and differential thermal analysis revealed that glycerol presence reduced the thermal stability, showing the influence of a plasticizer over the polymer structure. Water absorption capacity and contact angle were reduced with the increase of plasticizer concentration; on the other hand, the water vapor permeability increased, as expected, due to the hydrophilic nature of glycerol. The results indicated the potential application of C. hildmannianus mucilage as a promising material to produce edible films. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 45223.     

Impact of sweetness on the sensory acceptance of passion fruit nectar in Brazilian geographic regions

Passion fruit is the fourth most consumed fruit by the Brazilian population, especially in the form of juice, usually associated with the addition of sucrose. This study aimed to evaluate the ideal sucrose concentration, and its impact on the consumer's acceptance of passion fruit nectar, sweetened with non-caloric sweeteners as sucrose replacers in five Brazilian geographic regions. The acceptance scores evaluated by anova and Tukey's Honestly Significant Difference test indicated that sucralose-sweetened samples were the most accepted, with no differences from the sucrose-sweetened sample (P < 0.05), with a positive purchase intention in the five regions studied (>50%). However, differences in the acceptance scores were observed for the other sweeteners among the regions. Despite the difference in the sweetness perception among the Brazilian geographic regions, it may not be the only determining factor in the acceptance of passion fruit nectar sweetened with non-caloric sweeteners.     

Modelling sorption kinetic of sponge cake crumb added with milk syrup

Sponge cake is an ideal product to be soaked with liquids, due to its porous structure; baking process causes differences in crumb features through the cake; therefore, it is important to know how these variations can affect its sorption capacity. The aim of this work was to study the relation between crumb cake structure at different levels (bottom, centre and top) and sorption characteristics when adding milk syrup. The Peleg model was the best to fit sorption data (R² > 0.9), showing that the rate constant (k₁) diminished as the cell density (r = 0.920) and gelatinisation percentage (r = 0.890) decreased, while moisture (r = ?0.999) and a_w (r = ?0.994) increased; cell density only correlated with gelatinisation percentage (r = 0.997); micrographs showed that the sorption rate increased as more gelatinised starch and coagulated protein were found. To optimise the sorption process, the gelatinisation degree and crumb structure (cell density and types of pores) should be considered.     

Expression of IL-8, IL-6 and IL-1β in Tears as a Main Characteristic of the Immune Response in Human Microbial Keratitis

Corneal infections are frequent and potentially vision-threatening diseases, and despite the significance of the immunological response in animal models of microbial keratitis (MK), it remains unclear in humans. The aim of this study was to describe the cytokine profile of tears in patients with MK. Characteristics of ocular lesions such as size of the epithelial defect, stromal infiltration, and hypopyon were analyzed. Immunological evaluation included determination of interleukine (IL)-1β, IL-6, IL-8, IL-10, IL-12 and tumor necrosis factor (TNF)-α in tear samples obtained from infected eyes of 28 patients with MK and compared with their contralateral non-infected eyes. Additionally, frequency of CD4⁺, CD8⁺, CD19⁺ and CD3⁻CD56⁺ cells was also determined in peripheral blood mononuclear cells in patients with MK, and compared with 48 healthy controls. Non-significant differences were observed in the size of the epithelial defect, stromal infiltration, and hypopyon. Nevertheless, we found an immunological profile apparently related to MK etiology. IL-8 > IL-6 in patients with bacterial keratitis; IL-8 > IL-6 > IL-1β and increased frequency of circulating CD3⁻CD56⁺ NK cells in patients with gram-negative keratitis; and IL-8 = IL-6 > IL-1β in patients with fungal keratitis. Characterization of tear cytokines from patients with MK could aid our understanding of the immune pathophysiological mechanisms underlying corneal damage in humans.