Immunodetection of NUT Protein: Implementation, Indications, and Results in a Tertiary Reference Center.

The immunodetection of NUT protein is a reliable tool to identify NUT carcinoma, a rare and still underdiagnosed tumor entity. The technique was implemented in 2017 in our department, a tertiary reference center with a large recruitment in all tumor types, including head and neck and thoracic tumors. We evaluated its use over a 6-year period (2017-2022) to (a) describe the indications for the technique, (b) determine the number of NUT carcinomas detected and confirmed by Fluorescence in situ hybridization, and (c) describe briefly the characteristics of these tumors. Over the study period, 382 NUT immunodetections were performed; the annual number of requests varied from 45 to 83. All 21 pathologists of the department made at least one request (range: 1 to 94; annual mean: 18.2). 54.7% of immunodetections were performed for internal cases, 37% for cases submitted for consultation, and 8.3% for cases submitted for confirmation of a suspected diagnosis. The main indications were poorly differentiated tumors of the head and neck region (39%) and the thorax (19.6%), and difficult-to-classify soft tissue tumors (11.8%). Twelve cases of NUT carcinoma were detected by immunohistochemistry and confirmed by Fluorescence in situ hybridization. Seven were from the head and neck region (4.7% of the tumors tested), 4 from lung or mediastinum (5.3%), 1 from an unknown primary at the time of diagnosis. In conclusion, the implementation of NUT immunodetection in the daily workflow of a pathology department improves the detection of NUT carcinoma. This becomes essential with the emergence of potential targeted therapies.

In vitro antioxidant activity evaluation of pine nut peptides (Pinus koraiensis) fermented by Bacillus subtilis LS-45.

In this study, we utilized the remarkable capabilities of Bacillus subtilis ls-45 during the fermentation process to generate pine nut peptide. Through gene sequencing, we confirmed the proficiency of Bacillus subtilis ls-45 in producing protease, thereby serving as a valuable enzymatic source for protein hydrolysis. Our investigation focused on examining the variations in amino acid types and quantities between enzymatic pine nut protein peptide (EPP) and fermented pine nut protein polypeptide (FPP). Furthermore, we conducted a comprehensive assessment of the in vitro antioxidant activities of EPP and FPP, encompassing measurements of their Hydroxyl radical scavenging rate, Total reducing capacity, Superoxide anion scavenging rate, and ABTS+ radical scavenging rate. Notably, FPP exhibited superior antioxidant capacity compared to EPP. By employing semi-inhibitory mass concentration (IC50) analysis, we determined that FPP displayed enhanced efficacy in neutralizing hazardous free radicals when compared to EPP.

Believe It or 'Nut': Why It Is Time to Set the Record Straight on Nut Protein Quality: Pistachio (Pistacia vera L.) Focus.

There are growing public health movements to transition towards diets that are plant-based. However, confusion exists with concerns that plant-based proteins (including nuts) may be inferior with respect to protein quality. The present publication evaluates the evolution of protein quality concepts and explains the protein science related to pistachios. Pistachio nuts are a plant-based complete protein providing all nine EAAs in addition to an array of nutrients and phytochemicals. They have a PDCAAS of 73 and 81%, (raw and roasted pistachios, respectively), higher than that of many other tree nuts. From an environmental perspective transitioning towards plant-based diets (including nuts) could have potential to reduce total/green water footprints. Dietary guidelines are evolving yet nuts such as pistachios do not always have a clear place within these. Now appears to be a pertinent time to look at protein quality from the perspective of whole daily diets and dietary patterns, factoring in both health and environmental outcomes. Given updated modes of thinking, nuts such as pistachios have an important role to play in terms of providing ready-to-eat, good-quality, plant-based protein within daily diets.

Identification and Structure of Epitopes on Cashew Allergens Ana o 2 and Ana o 3 Using Phage Display.

BACKGROUND: Cashew (Anacardium occidentale L.) is a commercially important plant. Cashew nuts are a popular food source that belong to the tree nut family. Tree nuts are one of the eight major food allergens identified by the Food and Drug Administration in the USA. Allergies to cashew nuts cause severe and systemic immune reactions. Tree nut allergies are frequently fatal and are becoming more common. AIM: We aimed to identify the key allergenic epitopes of cashew nut proteins by correlating the phage display epitope prediction results with bioinformatics analysis. DESIGN: We predicted and experimentally confirmed cashew nut allergen antigenic peptides, which we named Ana o 2 (cupin superfamily) and Ana o 3 (prolamin superfamily). The Ana o 2 and Ana o 3 epitopes were predicted using DNAstar and PyMoL (incorporated in the Swiss-model package). The predicted weak and strong epitopes were synthesized as peptides. The related phage library was built. The peptides were also tested using phage display technology. The expressed antigens were tested and confirmed using microtiter plates coated with pooled human sera from patients with cashew nut allergies or healthy controls. RESULTS: The Ana o 2 epitopes were represented by four linear peptides, with the epitopes corresponding to amino acids 108-111, 113-119, 181-186, and 218-224. Furthermore, the identified Ana o 3 epitopes corresponding to amino acids 10-24, 13-27, 39-49, 66-70, 101-106, 107-114, and 115-122 were also screened out and chosen as the key allergenic epitopes. DISCUSSION: The Ana o 3 epitopes accounted for more than 40% of the total amino acid sequence of the protein; thus, Ana o 3 is potentially more allergenic than Ana o 2. CONCLUSIONS: The bioinformatic epitope prediction produced subpar results in this study. Furthermore, the phage display method was extremely effective in identifying the allergenic epitopes of cashew nut proteins. The key allergenic epitopes were chosen, providing important information for the study of cashew nut allergens.

Association Between the Seed Storage Proteins 2S Albumin and 11S Globulin and Severe Allergic Reaction After Flaxseed Intake.

BACKGROUND: Given the increased popularity of flaxseed in meals, several cases of allergy to these seeds have been reported. Little is known about the allergens implicated in hypersensitivity reactions to flaxseed. The present study aimed to identify the allergens involved in IgE-mediated reactions in 5 patients with a clinical history of severe systemic symptoms after flaxseed consumption. METHODS: Proteins that were potential allergens with IgE-binding capacity were purified from flaxseed extract using chromatography and identified via MALDI-TOF mass spectrometry. Immunoassays were performed using the 5 allergic patients' sera tested individually and as a pool. RESULTS: Immunoblotting of the flaxseed extract revealed a low-molecular-mass protein (around 13 kDa) in 4 of the 5 patients, while a protein of approximately 55 kDa was detected in 2 patients. The proteins were identified by mass spectrometry as flaxseed 2S albumin, which is included in the WHO/IUIS allergen nomenclature as Lin u 1, and 11S globulin. Inhibition assays revealed in vitro IgE-mediated cross-reactivity between Lin u 1 and peanut and cashew nut proteins, while IgE-mediated recognition of 11S globulin by patients' sera was partially inhibited by several plant-derived sources. CONCLUSIONS: Seed storage proteins from flaxseed were involved in the development of severe symptoms in the 5 patients studied and exhibited cross-reactivity with other allergenic sources. Besides the severity of flaxseed allergy in patients sensitized to 2S albumin, this is the first time that 11S globulin has been identified as a potential allergen. Taking these data into account should ensure a more accurate diagnosis.

Identification of nut protein-derived peptides against SARS-CoV-2 spike protein and main protease.

Recently, an outbreak of a novel coronavirus disease (COVID-19) has reached pandemic proportions, and there is an urgent need to develop nutritional supplements to assist with prevention, treatment, and recovery. In this study, SARS-CoV-2 inhibitory peptides were screened from nut proteins in silico, and binding affinities of the peptides to the SARS-CoV-2 main protease (Mpro) and the spike protein receptor-binding domain (RBD) were evaluated. Peptide NDQF from peanuts and peptide ASGCGDC from almonds were found to have a strong binding affinity for both targets of the coronavirus. The binding sites of the NDQF and ASGCGDC peptides are highly consistent with the Mpro inhibitor N3. In addition, NDQF and ASGCGDC exhibited an effective binding affinity for amino acid residues Tyr453 and Gln493 of the spike RBD. Molecular dynamics simulation further confirmed that the NDQF and ASGCGDC peptides could bind stably to the SARS-COV-2 Mpro and spike RBD. In summary, nut protein may be helpful as nutritional supplements for COVID-19 patients, and the screened peptides could be considered a potential lead compound for designing entry inhibitors against SARS-CoV-2.

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.

The necessity of enzymatically hydrolyzing walnut protein to exert antihypertensive activity based on in vitro simulated digestion and in vivo verification.

Since not all proteins are suitable for preparing bioactive peptides by enzymatic degradation, the purpose of this study is to evaluate the necessity of walnut protein (WP) enzymolysis to exert its potential antihypertensive activity. Five proteases were used to hydrolyze WP to produce WP hydrolysate (WPH) enzymatically. The angiotensin-I-converting enzyme (ACE) inhibitory activity of WP and WPH before and after simulated digestion in vitro was measured, and the antihypertensive effect was evaluated in vivo. The results showed that after simulated digestion in vitro, the ACE inhibitory activity of WP digests (44.85%) was not significantly different from that of WPH digests (p > 0.05). In vivo experimental results showed that both WP and WPH had significant blood pressure lowering effects in the acute and long-term administrative experiments. The mechanism of its antihypertensive activities was regulating the balance of the renin-angiotensin-aldosterone system and the kallikrein-kinin system by inhibiting ACE activities in tissues and regulating the level of endothelium-derived vasoconstrictor factors and relaxing factors in serum. It seems unnecessary to carry out enzymatic hydrolysis to produce walnut peptides with antihypertensive activity.

Consumo de frutos secos y función cognitiva: una revisión sistemática / Nut consumption and cognitive function: a systematic review

Los patrones dietéticos ricos en antioxidantes podrían contribuir a la prevención y el tratamiento de las fases previas de demencia. Los frutos secos tienen una apreciable carga antioxidante y hay evidencia de sus efectos beneficiosos sobre la incidencia de varias enfermedades crónicas y las tasas de mortalidad general. Además, son ricos en ácidos grasos polinsaturados, que también parecen desempeñar un papel positivo en la neurogénesis. El objetivo de esta revisión fue resumir la evidencia de los estudios relacionados con los efectos del consumo de frutos secos sobre la función cognitiva en personas adultas. Se realizó una búsqueda sistemática de artículos publicados en PubMed, Scopus y Web of Science. Un total de 19 artículos cumplieron los criterios de inclusión (siete transversales, cuatro de cohortes y ocho experimentales), que fueron extraídos y revisados de forma independiente por dos revisores. La evidencia proveniente de los estudios transversales y de cohortes resultó incierta, por la disparidad de resultados y su riesgo de sesgo. Sin embargo, en la mayoría de estudios experimentales se observó un efecto protector del consumo de frutos secos sobre alguna dimensión de la función cognitiva, y la calidad metodológica de estos fue aceptable. Además, los efectos parecen independientes del tipo de fruto seco, la cantidad ingerida, la edad y el estado basal de los consumidores. En resumen, estos resultados sugieren que incluir el consumo diario moderado de algún fruto seco en la dieta saludable de las personas adultas podría tener efectos beneficiosos sobre su función cognitiva. No obstante, son necesarios más estudios observacionales longitudinales y experimentales bien diseñados, que proporcionen firmeza a esta evidencia, hasta la fecha sugestiva y de una calidad moderadamente baja

Antioxidant-rich diet patterns could contribute to the prevention and treatment of early stages of dementia. Nuts have an appreciable antioxidant load and there is evidence of their positive effects on several chronic diseases incidence and death rates. Moreover, they are rich in polyunsaturated fatty acids, which might also play a positive role in neurogenesis. The aim of this systematic review was to summarize the evidence from studies related to the effects of nut consumption on cognitive function among adults. We conducted a systematic search of articles published in PubMed, Scopus and Web of Science. A total of 19 articles met the inclusion criteria (seven cross-sectional, four prospective cohorts and eight experimental); these were independently extracted and reviewed by two reviewers. The evidence from the cross-sectional and cohort studies was uncertain, due to the disparity of results and risk of bias. However, in most experimental studies a protective effect of nut consumption on some dimension of cognitive function was observed and the methodological quality of these studies was acceptable. In addition, the effects appear to be independent of nut type, amount of intake, age and baseline status of subjects. In summary, these results suggest that the inclusion of daily nut consumption in the healthy diet pattern of adults could have positive effects on their cognitive function. Nevertheless, more well-designed longitudinal and experimental studies are needed to provide strength to this suggestive evidence

Identification and characterization of major IgE binding of purified allergenic protein (11 kDa) from Buchanania lanzan.

Tree nut along with peanut are among the most potent food allergens, responsible for frequently inducing the IgE-mediated hypersensitivity reaction. Our aim was identification, purification of Buchanania lanzan (Bl-11 kDa) protein along with characterization and assessment of allergenic potential of clinically relevant allergen. Further study was executed in clinical samples of sensitive patients, BALB/c mice, and in-vitro. A major IgE binding 11-kDa protein from Buchanania lanzan was purified by anion exchange chromatography, reverse phase high pressure liquid chromatography (RP-HPLC) and characterized using peptide mass fingerprinting (PMF). Buchanania lanzan (Bl-11 kDa) protein shows the pepsin resistance and depicts IgE interacting capacity to Buchanania lanzan allergic patient's sera as well as sensitized mice sera. It also showed increase in the allergic mediator's like IgE, IgG1, histamine levels in sensitized mice sera. Further study was carried out in-vitro (RBL-2H3 cells) and increased release mast cell degranulation mediators such as ß-hexosaminidase, histamine, CysL and PGD2 in the culture supernatant was found. The activation of Th2 cytokines/transcription factors and expression of molecular markers in the downstream of mast cell signaling were up-regulated while the Th1 transcriptional factor (T-bet) was decreased in Bl-11 kDa protein treated mice. Conclusively, our study demonstrates Buchanania lanzan purified protein to be potential allergen that may generate an allergic reaction in sensitized individuals, and one of the most important IgE binding protein responsible for its allergenicity.

Antidiabetic effects of water-soluble Korean pine nut protein on type 2 diabetic mice.

Korean pine nut protein (PNP) has a variety of biological activities, which are good for human health, but its ability to preventing diabetes has not been reported. This study evaluated the effects of water-soluble proteins of Korean pine nut obtained from a dilute alkali extract on carbohydrate metabolism of type 2 diabetic mice on a model of diabetes induced using a high fat diet combined with streptozotocin. The results showed that the hypoglycemic effect of PNP at a middle dose was the most significant, which was 38.7% lower than that of control. The extract significantly improved the oral glucose tolerance and liver indexes, increased the activity of the carbohydrate metabolism enzymes, and regulated the expression of the function of key genes for carbohydrate metabolism. It had a positive effect on both insulin resistance and glycolytic/gluconeogenesis signaling. In conclusion, PNP can regulate fasting blood glucose, improve insulin resistance, correct the glucose metabolism disorder in diabetic mice, and have a positive regulatory role. As the functional food, it has the potential to be beneficial in the treatment of type 2 diabetes mellitus as a new hypoglycemic functional food.

The umami intensity enhancement of peanut protein isolate hydrolysate and its derived factions and peptides by Maillard reaction and the analysis of peptide (EP) Maillard products.

This study aimed to examine the feasibility of improving umami taste of peanut protein isolate hydrolysate (PPIH) and its fractions (obtained via ethanol precipitation and gel filtration chromatography) and peptide constituents, through their reactions with glucose at 98 °C for 70 min (Maillard reaction). To get insights into the characteristics of the peptide contributors with high umami intensity and/or umami-enhancing activity, Maillard reaction-guided and sensory-guided fractionation and characterization using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) and eXpose algorithm calculation method were subsequently involved. Three umami and umami-enhancing peptides, three umami-enhancing peptides with other tastes, three bitter peptides and one astringent peptide were found. For the first time, peptide TP was reported to suppress umami intensity of monosodium glutamate (MSG) aqueous solution. Combined treatments of enzymatic hydrolysis and Maillard reaction on PPI facilitated umami enhancement. Maillard products of identified peptides had higher umami intensities and/or greater umami-enhancing abilities. Pyroglutamylproline (pyroEP) and glucosylated EP were discovered as the major compounds of EP Maillard product.

Walnut oral immunotherapy for desensitisation of walnut and additional tree nut allergies (Nut CRACKER): a single-centre, prospective cohort study.

BACKGROUND: The safety and efficacy of oral immunotherapy for tree nut allergy has not been demonstrated to date, and its effectiveness is complicated by the high prevalence of co-allergies to several nuts. This study aimed to investigate the use of walnut oral immunotherapy in the desensitisation of walnut and additional tree nuts in patients who are co-allergic to several nuts. METHODS: In a single-centre, prospective cohort study (the Nut Co-Reactivity ACquiring Knowledge for Elimination Recommendations study) at the Institute of Allergy, Immunology, and Paediatric Pulmonology at the Yitzhak Shamir Medical Centre, we recruited patients aged 4 years or older who were allergic to walnut, with or without co-allergy to pecan, hazelnut, and cashew. The diagnosis of each food allergy was based on a positive skin prick test or specific serum IgE (≥0·35 kUA/L) to the corresponding nut together with a positive oral food challenge, unless an immediate (within 2 h of exposure) reaction in the past year had been documented. Patients with uncontrolled asthma or a medical contraindication to receive adrenaline were excluded. Patients were assigned to walnut oral immunotherapy or the control group (observation and strict dietary exclusion) on the basis of the order of presentation to the clinic. Oral immunotherapy began with a 4-day dose-escalation phase to establish the single highest tolerated dose, which was consumed daily at home for 24 days; subsequent monthly dose escalations were repeated until 4000 mg walnut protein was achieved. Patients who were desensitised to walnut continued to consume 1200 mg walnut protein daily for 6 months as maintenance. The primary outcome was walnut desensitisation (passing an oral food challenge with 4000 mg of walnut protein) at the end of the study, analysed by intention to treat. In patients who were co-allergic to pecan, hazelnut, and cashew, the proportion who achieved cross-desensitisation to these nuts in addition to walnut desensitisation was examined. FINDINGS: 73 patients with a walnut allergy were enrolled between May 15, 2016, and Jan 14, 2018. 49 (89%) of 55 patients in the oral immunotherapy group were desensitised to walnut compared with none of 18 patients in the control group (odds ratio 9·2, 95% CI 4·3-19·5; p<0·0001). Following walnut desensitisation, all patients who were co-allergic to pecan (n=46) were also desensitised to pecan. Additionally, 18 (60%) of 30 patients who were co-allergic to hazelnut or cashew, and 14 (93%) of 15 patients who were co-allergic to hazelnut alone, were either fully desensitised or responded to treatment. 47 (85%) of 55 patients had an adverse reaction (mostly grade 1 or 2) during up-dosing in the clinic; eight patients required intramuscular epinephrine in response to a dose at home. Of 45 patients who had follow-up data for the maintenance phase, all maintained walnut desensitisation and one patient required epinephrine during this period. INTERPRETATION: Walnut oral immunotherapy can induce desensitisation to walnut as well as cross-desensitisation to pecan and hazelnut in patients who have tree nut co-allergies, with a reasonable safety profile. A low daily dose of the allergen maintains desensitisation. FUNDING: None.

Mouse strain differences in response to oral immunotherapy for peanut allergy.

BACKGROUND: Promising therapies for food allergy are emerging, mostly based on animal experimentation. However, different mouse strains are used, which may make it hard to compare experiments. The current study investigated whether the immunological differences between C3H/HeOuJ (C3H) and BALB/c mice lead to differences in efficacy of peanut-specific immunotherapy. METHODS: After sensitization using peanut extract (PE), C3H and BALB/c mice received oral immunotherapy (OIT) by intragastric dosing for three weeks. Hereafter, mice were exposed to PE via the intradermal, intragastric and intraperitoneal route, to determine allergic outcomes. Furthermore, PE-specific antibody and cytokine production were determined and the number of various immune cells at different time points during the study were measured. RESULTS: OIT protected C3H mice against anaphylaxis, whereas no anaphylaxis was seen in BALB/c mice. In contrast, OIT induced an increase in MMCP-1 levels in BALB/c mice but not in C3H mice. No effect of OIT on the acute allergic skin response was observed in either strain. Specific antibody responses showed similar patterns in both strains for IgA and IgG1. IgE levels were a tenfold higher in BALB/c mice and after the intragastric challenge (day 70) OIT-treated BALB/c mice showed induced IgE levels. Moreover, in C3H mice IgG2a levels were higher and increased in response to OIT and challenges. After the final challenge, but not at other timepoints MLN-derived lymphocytes from OIT-treated BALB/c mice produced less IL-13 and IL-5 compared to control-treated mice, whereas no differences were seen in case of C3H mice. CONCLUSIONS: Taken together, these results show that the C3H strain is more suitable to study clinical outcomes of OIT, whereas the BALB/c strain is more optimal to study T cell responses.

Variability studies of allochthonous stone pine (Pinus pinea L.) plantations in Chile through nut protein profiling.

Stone pine (Pinus pinea) is characterized by low differentiation of growth parameters, high phenotypic plasticity and low genetic variability; detecting its diversity in introduced Chilean populations is therefore relevant for conservation and breeding programs. Here, variability among allochthonous Stone pine populations in Chile was explored using electrophoresis-based proteomic analysis of pine nuts. Cones from 30 populations distributed along a climatic gradient in Chile were surveyed and sampled, and proteins were extracted from seed flour using the TCA-acetone precipitation protocol. Extracts were subjected to SDS-PAGE and 2-DE for protein resolution, gel images captured, and spot or bands intensity quantified and subjected to statistical analysis (ANOVA, unsupervised Hierarchical Analysis Clustering and PLS regression). Protein yield ranged among populations from 161.7 (North populations) to 298.7 (South populations) mg/g dry weight. A total of 50 bands were resolved by SDS-PAGE in the 6.5-200 kDa Mr. range, of which 17 showed quantitative or qualitative differences, with 12 proteins identified. Pine nut extracts from the most distant populations were analyzed by 2-DE and a total of 129 differential spots were observed, out of which 13 were proposed as putative protein markers of variability. Out of the 129 spots, 118 proteins were identified after MALDI-TOF/TOF analysis. Identified proteins were classified into two principal categories: reserve and stress related. We provide the first protein map of P. pinea nuts. The use of a proteomic approach was useful to detect variability of Stone pine across three Chilean macrozones, with correlations between protein profiles and geoclimatic parameters, suggesting a new approach to study the variability of this species. BIOLOGICAL SIGNIFICANCE: This study presents the first protein map of Stone pine nuts, relevant for the advancement of protein characterization in pine nuts. Putative protein markers are proposed, evidencing that a proteomic approach may be useful to detect variability of Stone pine across Chilean macrozones, suggesting a new approach to study the variability of this species, which may also be extrapolated to other forest fruit species.