Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen Gene Ara h 2

The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR–Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR–Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR–Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.

Trace Elements Analysis of Tunisian and European Extra Virgin Olive Oils by ICP-MS and Chemometrics for Geographical Discrimination

The aim of this study was to investigate the levels of trace elements in olive oils from different locations and their use for geographical authentication. Concentrations of seventeen elements were determined in a total of 42 olive oils from Tunisia, Spain (Basque country), and southern France, and in nine soil samples from Tunisia by quadrupole inductively plasma mass spectrometry. The compilation of appropriate techniques integrated into the analytical procedure achieved a precision (RSD) between 2% and 15% and low limits of detection (between 0.0002 and 0.313 µg kg−1). The accuracy of the analytical method applied for olive oil analysis was evaluated using SRM NIST 2387 Peanut butter. The recoveries obtained after microwave-assisted digestion for the certified elements ranged between 86% and 102%. Concentrations of non-certified elements (V, Cr, Co, Ni, Ba, Rb, Sr, Cd, Pb, and As) were presented. The use of Pearson correlation applied on paired Tunisian oil/soil samples has shown that several elements (Mg, Mn, Ni, and Sr) were significantly correlated. The multivariate statistics using principal component analysis have successfully discriminated against three studied origins. The most significant variables were the elemental concentrations of Cu, Cr, Fe, Mn, Sr, V, and Zn. This study shows the potential of applying trace elements profiles for olive oil geographical discrimination.

Valorization of the Olive Oil Production Residue: Healthy Ingredient for Developing High Value-Added Spread

This study focused on the development of a new olive-based product enriched with proteins, antioxidant compounds and functional properties, aiming at valorization of the olive residue from the typical olive oil extraction process. Olive oil was extracted using a patented cold pressing machine, resulting in a valuable source of bioactive compounds in the form of olive residue (pulp ~70%, pit- and skin-free). Conventional (solid: water ratio 1:4, 25 °C, 2 h) and HP-assisted (200 MPa, solid:water ratio 1/4, 10 min) de-bittering processes were carried out in order to remove oleuropein content (up to 50%) from olive residue, thus decreasing its bitter taste. The olive spread was prepared with olive residue (50%) and honey (25%), enriched with proteins (24.5%) from nuts. Quality parameters, nutritional attributes, microbiological analysis, phenolic compounds and oxidative stability as well as sensory evaluation of the olive spread were conducted during storage. Its shelf-life was estimated (prediction through accelerated tests) as 9.5 months when stored at 4 °C. The organoleptic evaluation showed that the proposed olive spread had a texture similar to peanut butter and could potentially be consumed as a health-promoting spread due to its high content of proteins (~10 g/100 g), crude fibers (>6 g/100 g) and essential fatty acids.

Paeonol Disrupts the Integrity of Aspergillus flavus Cell Walls via Releasing Surface Proteins, Inhibiting the Biosynthesis of β-1,3-Glucan and Promoting the Degradation of Chitin, and an Identification of Cell Surface Proteins

Paeonol can effectively inhibit Aspergillus flavus (A. flavus) via damaging cell walls. In this work, paeonol treatment remarkably destroyed both the outer amorphous layer and the inner fibrous layer of cell walls. Furthermore, FT-IR and XPS characterization showed that OH functional groups were altered and proteins in the outer layer were released. According to proteomic analysis, 605 proteins have been identified and annotated. The activities of β-1,3-glucan synthase and chitinase were prohibited and promoted, respectively, by paeonol treatment, however, the activities of β-1,3-glucanase and chitin synthase were not influenced. QRT-PCR results suggested that FKSP, CHIIII, and CHIV genes might be the antifungal targets of paeonol. In addition, paeonol can effectively restrain the pathogenicity of A. flavus on peanut butter. This study provided a new elucidation on the mode of action of paeonol against cell walls of A. flavus, facilitating the application of paeonol in the preservation of agricultural products.

Assessing the Attractiveness of Three Baits for Roof Rats in California Citrus Orchards

Roof rats (Rattus rattus) are common pests of tree fruit and nut orchards in California. Tracking tunnels are an effective and practical tool for monitoring changes in roof rat numbers in orchard systems, but they rely on the use of attractive baits to draw in rats to the tracking surface. The identification of baits that are both attractive and readily available would be of great assistance for monitoring changes in roof rat numbers within agricultural fields. Therefore, we tested the attractiveness of a soft bait (Liphatech Rat and Mouse AttractantTM), a wax block (Liphatech NoToxTM), peanut butter, and a control (i.e., no bait applied) to roof rats when applied within tracking tunnels. We did not observe a difference in visitation to any of the tested attractants, although all attractants yielded greater visitation rates than control tunnels. However, tracking tunnels without any attractant were still visited at a relatively high rate, indicating the tracking tunnel is attractive to roof rats. As such, monitoring tools that provide shelter may increase detectability, although this would need to be verified. Regardless, all three food-based lures we tested increased the attractiveness of tracking tunnels and could be used to increase visitation to these monitoring devices.

Development of an energy-dense biscuit suitable for primary school learners for the South African National School Nutrition Programme

South Africa has a well established National School Nutrition Programme (NSNP). Despite rescheduling the mealtime to two hours after the start of the school day to accommodate learners who come to school on an empty stomach, a gap still exists as many children start their learning day with low energy and concentration levels. A cost-effective, energy-dense snack served at the start of the school day can be a solution to sustain learners until the main meal is served. Cross-sectional surveys were used to determine the snack preferences of children and the product development process was used to develop a suitable snack. An energy-dense peanut butter biscuit was developed based on those surveys as well as a scoping review of previous snack studies. The energy-dense developed product provided 1388kJ (61.0%) of energy from fat, 688kJ (30.2%) of energy from carbohydrates and 201kJ (8.8%) of energy from protein per 100g. The biscuit conformed to microbial testing standards. Shelf-life analysis projected a shelf-life of five weeks fresh and five months in food grade packaging. Sensory results showed that there was no significant difference in sensory scores across gender (p=0.691) and age (p=0.706). More of the learners (n=56, 69.1%) found the biscuit to be ‘Super good’ than the other ratings (p<.0005). When compared with similar biscuit products currently on the market, it was found that the developed biscuit was the most reasonably priced. The developed biscuit has the potential to serve as a solution to hidden hunger for children that come to school on an empty stomach. This versatile snack solution has potential for continuity of use even during periods of national crisis as with COVID-19, when learners’ nutritional needs may be most vulnerable.

Fatal anaphylaxis due to peanut exposure from oral intercourse

Background Intimacy-related allergic reactions, including anaphylaxis, are under-reported due to social stigma, lack of awareness, and misdiagnosis. The differential diagnosis for intimacy-related anaphylaxis is extensive and includes systemic human seminal plasma allergy, exercise-induced anaphylaxis, asthma exacerbation, latex allergy, and transference of food or drug allergens through saliva or seminal fluid. Case presentation Two adolescents met on a popular dating phone application. One individual had a long-standing history of asthma and peanut allergy. Although they never kissed, the male with peanut allergy received fellatio, while the other male had eaten peanut butter before they met. During fellatio, the peanut allergic male developed respiratory symptoms, used his bronchodilator, and collapsed. He remained unconscious despite aggressive interventions by emergency personnel called to the site. The clinical history and autopsy results suggested anaphylaxis to peanut allergen exposure from the intimate exposure as the cause of death. Discussion and conclusions To date, nearly all reported cases of intimacy-related anaphylaxis involve symptomatic women. This is the first report of intimacy-related anaphylaxis involving men who have sex with men and the first report of potential allergen transfer from oral mucosa to a patient receiving fellatio. Based on the paucity of published cases, death from intimacy-related anaphylaxis is exceedingly rare. Post-mortem analysis is inherently difficult, as an elevated tryptase level has myriad potential causes; nevertheless, the authors suggest that intimacy-related anaphylaxis due to peanut allergy is the most likely diagnosis. With increasing popularity of relationship applications, especially amongst stigmatized populations, this case highlights the importance of allergy awareness and patient education to decrease risk, particularly in the adolescent population, who are already at increased risk of severe anaphylaxis. Especially amongst those participating in intimate activities, disclosure of one’s allergies warrants discussion, as the outcome can be fatal. Our case demonstrates the crucial need for increased advocacy in food allergy, education around intimacy-related anaphylaxis, and the importance of allergy awareness and prevention across all populations.