New Frontiers: Precise Editing of Allergen Genes Using CRISPR

Genome engineering with clustered regularly interspaced short palindromic repeats (CRISPR) technology offers the unique potential for unequivocally deleting allergen genes at the source. Compared to prior gene editing approaches, CRISPR boasts substantial improvements in editing efficiency, throughput, and precision. CRISPR has demonstrated success in several clinical applications such as sickle cell disease and β-thalassemia, and preliminary knockout studies of allergenic proteins using CRISPR editing show promise. Given the advantages of CRISPR, as well as specific DNA targets in the allergen genes, CRISPR gene editing is a viable approach for tackling allergy, which may lead to significant disease improvement. This review will highlight recent applications of CRISPR editing of allergens, particularly cat allergen Fel d 1, and will discuss the advantages and limitations of this approach compared to existing treatment options.

ADJUSTING THE LOW MOLECULAR WEIGHT OF HYDROXYETHYL CELLULOSE AND APPLIED TO EXTRACT PROTEINS IN NATURAL RUBBER LATEX

Natural rubber latex (NRL) with “low protein content” is regarded as less allergenic latex for medical gloves, medical products, condoms, etc. Therefore, this study was conducted to strengthen the fundamental approach of making “low protein NRL” via oligosaccharide hydroxyl ethyl cellulose (oligoHEC) treatment. OligoHEC (Mw ~10,000 g/mol) is degradation product of HEC (Mw ~90,000 g/mol) by 1.5% H2O2 treatment combined with hydrothermal at temperature for 30 minutes at 121°C, pressurized rate of 0.38 mPa by autoclave equipment. OligoHECs were employed to form electrical bonds with the proteins. The target of this study is protein content of NRL ≤ 50 µg/g of rubber, we surveyed the effect of oligoHEC treatment towards the solution proteins content of NRL. Results show that oligoHEC at low concentration (0.25%) effectively extracted the proteins molecules. Interestingly, allergenic proteins content of NRL serum was decreased proportionally (< 50 µg/g of rubber) with the upsurge of oligoHEC concentration, suggesting deactivation of allergenic. These preliminary results indicate a potential approach to produce low allergenic risk NRL products with adding of oligoHEC

Precision Medicine in Mite Allergic Rhinitis

It is well-known that a correct diagnosis is necessary for effective treatment. In the case of allergic rhinitis due to mites, imprecise diagnosis with effective but improvable methods means that in many cases an optimal result is not reached in patients. The diagnosis of allergic rhinitis due to mite sensitization have to require more homogeneously reproducible diagnostic tests that try to encompass many more of the protein antigens contained in them. With the few proteins that the problem has usually focused on, there is no they would cover many of the clinically relevant allergens in a large proportion of patients. In this mini-review we try to highlight the importance of having good allergenic sources and briefly gather information on various allergenic proteins included in mites that could be clinically relevant. All this to try to get closer to a more accurate diagnosis. We are also talking about two diagnostic tools that are clearly out of use and that should be promoted in the consultations to obtain an even greater and better outcome in patients.

Evaluation of antigenicity and nutritional properties of enzymatically hydrolyzed cow milk

While enzymatic hydrolysis is an effective method for lowering the antigenicity of cow milk (CM), research regarding the antigenicity and nutritional traits of CM hydrolysate is limited. Here, we evaluated the protein content, amino acid composition, sensory traits, color, flow behavior, and antigenicity of CM following enzymatic hydrolysis. The results showed that enzymatic hydrolysis increased the degree of hydrolysis, destroyed allergenic proteins, including casein, β-lactoglobulin, and ɑ-lactalbumin, and significantly increased the content of free amino acids and nutritional quality. In particular, the antigenicity of CM was significantly reduced from 44.05 to 86.55% (P < 0.5). Simultaneously, the taste, color, and flow behavior of CM were altered, the sweetness and richness intensity decreased significantly (P < 0.5), and astringency and bitterness were produced. A slightly darker and more yellow color was observed in CM hydrolysate. In addition, apparent viscosity decreased and shear stress significantly increased with increasing shear rate intensity. The results will provide a solid theoretical foundation for the development of high-quality hypoallergenic dairy products.

AllerStat: Finding Statistically Significant Allergen-Specific Patterns in Protein Sequences by Machine Learning

Cutting-edge technologies such as genome editing and synthetic biology allow us to produce novel foods and functional proteins. However, their toxicity and allergenicity must be accurately evaluated. Allergic reactions are caused by specific amino-acid sequences in proteins (Allergen Specific Patterns, ASPs), of which, many remain undiscovered. In this study, we introduce a data-driven approach and a machine-learning (ML) method to find undiscovered ASPs. The proposed method enables an exhaustive search for amino-acid subsequences whose frequencies are statistically significantly higher in allergenic proteins. As a proof-of-concept (PoC), we created a database containing 21,154 proteins of which the presence or absence of allergic reactions are already known, and the proposed method was applied to the database. The detected ASPs in the PoC study were consistent with known biological findings, and the allergenicity prediction accuracy using the detected ASPs was higher than extant approaches.

Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS

The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0–15 kDa, 15–35 kDa, 35–70 kDa and 70–250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts.

Soybean and Lupine Addition in Hen Nutrition—Influence on Egg Immunoreactivity

Modifying hen fodder is a common way of changing eggs composition today. However, there is no information on the effect of the source of protein in the fodder replacement on egg allergenicity. This research aimed to detect potential differences in the immunoreactivity and protein composition of eggs from hens fed with fodder containing legume. The aim of the first step of the study was to select the proper solvent for extracting allergenic proteins from hen eggs. Two of them (containing Tween 20 and Triton 100) were selected, based on protein profile and concentration analysis. Egg-white- and egg-yolk-proteins extracts prepared with them were checked for potential differences, using SDS-PAGE electrophoresis, and then the Western-blot method, using sera from children allergic to eggs and soy. Preliminary studies on the influence of fodder composition on the composition of egg proteins suggest that the addition of soy and lupine to fodder modifies the expression of egg proteins. The observed differences in the immunoreactivity of proteins contained in hen egg-white samples do not seem to be as significant as the appearance of protein with a molecular weight of ~13 kDa in the yolk of eggs obtained from soybean-fed hens. This protein may increase the immunoreactivity of eggs for children allergic solely to soy.