Isolation,cloning, and characterization of the 2S albumin: A new allergen from hazelnut

Scope: 2S albumins are the major allergens involved in severe food allergy to nuts, seeds, and legumes. We aimed to isolate, clone, and express 2S albumin from hazelnut and determine its allergenicity. Methods: 2S albumin from hazelnut extract was purified using size exclusion chromatography and RP‐HPLC. After N‐terminal sequencing, degenerated and poly‐d(T) primers were used to clone the 2S albumin sequence from hazelnut cDNA. After expression in Escherichia coli and affinity purification, IgE reactivity was evaluated by Immunoblot/ImmunoCAP (inhibition) analyses using sera of nut‐allergic patients. Results: N‐terminal sequencing of a ～10 kDa peak from size exclusion chromatography/RP‐HPLC gave two sequences highly homologous to pecan 2S albumin, an 11 amino acid (aa) N‐terminal and a 10aa internal peptide. The obtained clone (441 bp) encoded a 147aa hazelnut 2S albumin consisting of a putative signal peptide (22 aa), a linker peptide (20 aa), and the mature protein sequence (105 aa). The latter was successfully expressed in E. coli. Both recombinant and natural 2S albumin demonstrated similar IgE reactivity in Immunoblot/ImmunoCAP (inhibition) analyses. Conclusion: We confirmed the postulated role of hazelnut 2S albumin as an allergen. The availability of recombinant molecules will allow establishing the importance of hazelnut 2S albumin for hazelnut allergy.     

Validating allergen coding genes (Cor a 1, Cor a 8, Cor a 14) as target sequences for hazelnut detection via Real-Time PCR

Hazelnuts have been shown to contain different allergenic proteins. Amongst these, major allergens Cor a 1 and Cor a 8 and the 2S albumin Cor a 14 were selected as targets to comparatively validate three Real-Time PCR protocols. We investigated both on the choice of the amplification target, and on the matrix effect on different sample foods. Applying statistics on the validation parameters obtained from the three protocols, we showed a significant difference in Ct values. This could turn critical when a high sensitivity method is required for the detection of hazelnut traces, confirming how fundamental the choice of the template during primer design phase is. Concluding, statistical approach represents a useful tool for the identification of the best performing primer pairs in Real-Time PCR. Cor a 8 gene permitted the identification of hazelnut based ingredients in complex foods, providing a significantly higher sensitivity in the PCR amplification, when compared to Cor a 1 and Cor a 14.     

Hazard assessment of edible oil refining: Formation of flammable mixtures in storage tanks

This work presents a methodology for the assessment of hazards due to the formation of flammable mixtures in edible oil refineries, with particular focus on storage tanks. Safety issues in these plants are due to the presence of residual volatile flammable solvents deriving from the extraction processes, which may accumulate in the storage tanks and generate flammable vapour mixtures. The methodology, based on the application of a thermodynamic model for the estimation of vapour phase composition in oil storage tanks as a function of operating conditions (i.e. temperature and residual solvent content), allows the identification of potential hazards due to formation of flammable mixtures inside the tanks. The model was first validated using available experimental data and then extended to case-studies representative of state-of-the-art industrial storage applications. The results of the case-studies were then used to calculate specific hazard indexes, providing a quick tool for preliminary assessment of hazards due to the formation of flammable mixtures in edible oil storage plants.     

Staphylococcus aureus Exfoliative Toxin E,Oligomeric State and Flip of P186: Implications for Its Action Mechanism

Staphylococcal exfoliative toxins (ETs) are glutamyl endopeptidases that specifically cleave the Glu381-Gly382 bond in the ectodomains of desmoglein 1 (Dsg1) via complex action mechanisms. To date, four ETs have been identified in different Staphylococcus aureus strains and ETE is the most recently characterized. The unusual properties of ETs have been attributed to a unique structural feature, i.e., the 180° flip of the carbonyl oxygen (O) of the nonconserved residue 192/186 (ETA/ETE numbering), not conducive to the oxyanion hole formation. We report the crystal structure of ETE determined at 1.61 Å resolution, in which P186(O) adopts two conformations displaying a 180° rotation. This finding, together with free energy calculations, supports the existence of a dynamic transition between the conformations under the tested conditions. Moreover, enzymatic assays showed no significant differences in the esterolytic efficiency of ETE and ETE/P186G, a mutant predicted to possess a functional oxyanion hole, thus downplaying the influence of the flip on the activity. Finally, we observed the formation of ETE homodimers in solution and the predicted homodimeric structure revealed the participation of a characteristic nonconserved loop in the interface and the partial occlusion of the protein active site, suggesting that monomerization is required for enzymatic activity.     

Does carbon nanopowder threaten amphibian development?

Lethal and teratogenic potentials of carbon nanoparticles (CNPs) in their amorphous form were investigated by the standardized Frog Embryo Teratogenesis Assay-Xenopus (FETAX), a 96-h in vitro whole-embryo toxicity test based on the amphibian Xenopus laevis. Embryos were acutely exposed to 1, 10, 100 and 500 mg/L CNP suspensions and evaluated for lethality, malformations and growth inhibition. Larvae were processed for histological and ultrastructural analyses to detect the main affected organs, to look for specific lesions at the subcellular level and to image and track CNPs into tissues. Only the highest CNP suspension resulted in being embryolethal for X. laevis larvae, while malformed larva percentages significantly differed from controls starting from 100 mg/L. The stomach and gut were the preferential CNP accumulation sites, on the contrary, the digestive epithelium remained intact. The analyses showed the presence of isolated nanoparticles and/or aggregates in different secondary target organs. CNPs were found in circulating erythrocytes. The research confirms the good tolerance of X. laevis towards pure elemental carbon in its nanoparticulate amorphous form, but highlights the possibility of CNP transfer toward all body areas.     

Kinetics of Soybean Oil Hydrolysis on Niobium Catalysts

The catalytic hydrolysis of soybean oil was used as an alternative for the production of monoglycerides (MG) and diglycerides (DG). The reactions were conducted in a stainless-steel tubular reactor in the temperature range of 240–290 °C, on niobium phosphate (NBP) and niobium oxide (NBO) as catalysts. In the hydrolysis reactions at 270 °C, the maximum selectivities of the products of interest were obtained at 22 % MG and 48 % DG for the reaction with NBP, and 7 % MG and 33 % DG with NBO, for 59 % and 36 % of triglyceride conversion in 10 min, respectively. The proposed kinetic model presented a good fit of the theoretical model with the experimental data, showing that the previous hypotheses considered for the mechanism development are suitable for describing the kinetics of soybean oil hydrolysis.     

Effect of stereoisomerism of poly(lactic acid) during neural guide conduit membrane synthesis

Poly(lactic acid) membranes are being developed as biomaterials for several purposes such as artificial implants for peripheral nerve injury, also known as neural guide conduits (NGC). These membranes need to meet standards of mechanical, degradability, and permeability properties, besides dimensional and structural requirements. Among the stereoisomers of polylactides, poly(l ‐lactic acid), and poly(d ,l ‐lactic acid) are the most used as biomaterials, having significant differences in solubility, crystallinity thermal, and mechanical properties. In this work, PLLA and PDLLA were compared for hollow fiber membrane synthesis by liquid induced phase separation. PLLA samples presented 18% of crystallinity, while PDLLA is amorphous. PDLLA and PLLA polymer solutions on N‐methyl‐pyrrolidone presented values of 3428 and 320.2 cP, respectively. In immersion of PLA‐NMP solutions in water, PLLA solution presented instantaneous demixing, while PDLLA showed a 28 s delay on precipitation. The PLLA–NMP–water has a larger demixing region compared to PDLLA–NMP–water system. Hollow fibers of both polymers presented closed external surface with finger‐like macropores morphology in their cross‐sections. PDLLA presented typical liquid–liquid demixing pores while PLLA presented spherulitical crystalline solid–liquid separation structures, which deeply compromised its mechanical properties. PDLLA presented as a good candidate for hollow fiber NGC material, as presented good mechanical resistance in tensile and suture simulating tests. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46190.     

Production,nutrient cycling and soil compaction to grazing of grass companion cropping with corn and soybean

Agricultural management systems are needed to simultaneously enhance production, promote plant diversity, improve nutrient cycling and reduce soil compaction. We investigated the effects of intercropped forage grass on production of corn (Zea mays L.) harvested for silage at 0.20 and 0.45 m height in the summer, as well as on production of subsequent forage, soybean [Glycine max (L.) Merr.] harvested for silage, nutrient cycling and soil responses on a Typic Haplorthox in Botucatu, São Paulo State, Brazil. Palisade grass cv. BRS Piatã [Urochloa brizantha cv. BRS Piatã] was the introduced companion crop with corn (Years 1 and 2), while signal grass [Urochloa decumbens cv. Basilisk] was the residual weedy species in comparison. Guineagrass cv. Aruãna [Megathyrsus maximus cv. Aruãna] was the introduced companion crop with soybean (Year 3), with only a residual effect of crop systems from the previous two years. After the corn silage harvest, pasture was grazed by lambs in winter/spring using a semi-feedlot system. When cut at 0.45 m compared with 0.20 m height, corn intercropped with palisade grass had greater leaf nutrient concentration, improved agronomic characteristics, forage mass of pasture for grazing by lambs, greater surface mulch produced, and greater quantity of N, P and K returned to soil. Greater soil organic matter, P, K and Mg concentration, and base saturation in the surface soil depth and lower soil penetration resistance at all depths occurred at 0.45 m than at 0.20 m corn silage cutting height intercropped with palisade grass. Analyzing the system as a whole, harvesting corn silage crop with palisade grass intercrop at 0.45 m height was the most viable option in this integrated crop-livestock system.