Nonspecific Lipid Transfer Protein Research Articles

Proteomic characterization and cytotoxic potential of proteins from Cuscuta (Cuscuta epithymum (L.) crude herbal product against MCF-7 human breast cancer cell line

BackgroundThe burden of breast cancer, the second leading cause of death worldwide, is increasing at an alarming rate. Cuscuta, used in traditional medicine for different ailments, including cancer, is known for containing phytochemicals that exhibit anticancer activity; however, the bioactivities of proteins from this plant remain unexplored. This study aimed to screen the cytotoxic potential of proteins from the crude herbal product of Cuscuta epithymum(L.) (CE) harvested from the host plants Alhagi maurorum and Medicago sativa.MethodsThe proteins from CE were extracted using a salting-out method, followed by fractionation with a gel filtration chromatography column. Gel-free shotgun proteomics was subsequently performed for protein characterization. The viability assay using MTT was applied to deduce the cytotoxic potential of proteins against MCF-7 breast cancer cells, with further exploration of the effect of treatment on the expression of the apoptotic mediator BCL2-associated X protein (BAX) and B-cell lymphoma protein 2 (BCL-2) proteins, using western blotting to strengthen the findings from the in vitro viability assay.ResultsThe crude proteins (CP) of CE were separated into four protein peaks (P1, P2, P3, and P4) by gel filtration chromatography. The evaluation of potency showed a dose-dependent decline in the MCF-7 cell line after CP, P1, P2, and P3 treatment with the respective IC50 values of 33.8, 43.1, 34.5, and 28.6 µg/ml. The percent viability of the cells decreased significantly upon treatment with 50 µg/ml CP, P1, P2, and P3 (P < 0.001). Western-blot analysis revealed upregulation of proapoptotic protein BAX in the cells treated with CP, P3 (P < 0.01), and P2 (P < 0.05); however, the antiapoptotic protein, BCL-2 was downregulated in the cells treated with CP and P3 (P < 0.01), but no significant change was detected in P2 treated cells. The observed cytotoxic effects of proteins in the CP, P1, P2, and P3 from the in vitro viability assay and western blot depicted the bioactivity potential of CE proteins. The database search revealed the identities of functionally important proteins, including nonspecific lipid transfer protein, superoxide dismutase, carboxypeptidase, RNase H domain containing protein, and polyribonucleotide nucleotidyltransferase, which have been previously reported from other plants to exhibit anticancer activity.ConclusionThis study indicated the cytotoxic activity of Cuscuta proteins against breast cancer MCF-7 cells and will be utilized for future investigations on the mechanistic effect of active proteins. The survey of CE proteins provided substantial data to encourage further exploration of biological activities exhibited by proteins in Cuscuta.

Oral allergy syndrome to kohlrabi in a patient with IgE sensitization to a weed pollen non-specific lipid transfer protein

Oral allergy syndrome to kohlrabi in a patient with IgE sensitization to a weed pollen non-specific lipid transfer protein

Group 2 innate lymphoid cells are key in lipid transfer protein allergy pathogenesis.

Immunopathology in food allergy is characterized by an uncontrolled type 2 immune response and specific-IgE production. Recent studies have determined that group 2 innate lymphoid cells (ILC2) participate in the food allergy pathogenic mechanism and their severity. Our objective was to investigate the role of ILC2 in peach-allergic patients due to non-specific lipid transfer protein (Pru p 3) sensitization. The immune response in peripheral blood mononuclear cells was characterized in lipid transfer protein-allergic patients and healthy controls. We have analyzed the Pru p 3 uptake on ILC2, the expression of costimulatory molecules, and their involvement on the T-cell proliferative response and cytokine production under different experimental conditions: cytokines involved in group 2 innate lymphoid cell activation (IL-33 and IL-25), Pru p 3 as main food allergen, and the combination of both components (IL-33/IL-25+Pru p 3) using cell sorting, EliSpot, flow cytometry, and confocal microscopy. Our results show that Pru p 3 allergen is taken up by group 2 innate lymphoid cells, regulating their costimulatory molecule expression (CD83 and HLA-DR) depending on the presence of Pru p 3 and its combination with IL-33/IL-25. The Pru p 3-stimulated ILC2 induced specific GATA3+Th2 proliferation and cytokine (IL-4, IL-5, and IL-13) production in lipid transfer protein-allergic patients in a cell contact-dependent manner with no changes in Tbet+Th1- and FOXP3+Treg cell differentiation. The results indicate that in lipid transfer protein-allergic patients, the responsible allergen, Pru p 3, interacts with group 2 innate lymphoid cells, promoting a Th2 cell response. Our results might be of interest in vivo, as they show a role of group 2 innate lymphoid cells as antigen-presenting cells, contributing to the development of food allergy. Consequently, group 2 innate lymphoid cells may be considered as potential therapeutic targets.

Applying a non-GMO breeding approach with an identified natural variation to reduce food allergen Len c3 in Lens culinaris seeds.

Lentils (Lens culinaris) are produced in diverse agroecological regions and are consumed as one of the most important food legumes worldwide. Lentils possess a nutritional profile from a human health perspective that is not only nutrient dense but also offers a better balance between protein and carbohydrates. However, lentil causes food allergy, which has been a significant concern due to increased consumption in parts of the world. Len c3, a non-specific lipid transfer protein (LTP), was identified as one of the allergens in lentil seeds. In this study, we identified an LTP gene Lcu.2RBY.4g013600 that encodes the lentil allergen Len c3. We then focused on gene screening from a collection of natural accessions to search for natural mutations of the Len c3 allergen-encoding gene. A natural lentil line M11 was identified with mutations at LcLTP3b and low accumulation of vicilin through genomic-assisted approaches. Furthermore, we generated a pool of lentil germplasms with LcLTP3b mutation background through crossing the identified lentil plant M11 with two lentil cultivars, CDC Redmoon and CDC Gold. These generated lentil hybrids can be used as a breeding resource targeting at reducing allergen risk in lentil consumption.

Genome-wide identification and evolutionary analysis of the AP2/EREBP, COX and LTP genes in Zea mays L. under drought stress

AP2 (APETALA2)/EREBP (ethylene-responsive element-binding protein), cytochrome c oxidase (COX) and nonspecific lipid transfer proteins (LTP) play important roles in the response to drought stress. This is the first study to identify the COX gene in Zea mays L. via genome-wide analysis. The qRT‒PCR results indicated that AP2/EREBP, COX and LTP were downregulated, with fold changes of 0.84, 0.53 and 0.31, respectively, after 12 h of drought stress. Genome-wide analysis identified 78 AP2/EREBP, 6 COX and 10 LTP genes in Z. mays L. Domain analysis confirmed the presence of the AP2 domain, Cyt_c_Oxidase_Vb domain and nsLTP1 in the AP2/EREBP, COX and LTP proteins, respectively. The AP2/EREBP protein family (AP2) includes five different domain types: the AP2/ERF domain, the EREBP-like factor (EREBP), the ethylene responsive factor (ERF), the dehydration responsive element binding protein (DREB) and the SHN SHINE. Synteny analysis of the AP2/EREBP, COX and LTP genes revealed collinearity orthologous relationships in O. sativa, H. vulgare and A. thaliana. AP2/EREBP genes were found on the 10 chromosomes of Z. mays L. COX genes were found on chromosomes 1, 3, 4, 5, 7 and 8. LTP genes were found on chromosomes 1, 3, 6, 8, 9 and 10. In the present study, the Ka/Ks ratios of the AP2/EREBP paralogous pairs indicated that the AP2/EREBP genes were influenced primarily by purifying selection, which indicated that the AP2/EREBP genes received strong environmental pressure during evolution. The Ka/Ks ratios of the COX-3/COX-4 paralogous pairs indicate that the COX-3/COX-4 genes were influenced primarily by Darwinian selection (driving change). For the LTP genes, the Ka/Ks ratios of the LTP-1/LTP-10, LTP-5/LTP-3 and LTP-4/LTP-8 paralogous pairs indicate that these genes were influenced primarily by purifying selection, while the Ka/Ks ratios of the LTP-2/LTP-6 paralogous pairs indicate that these genes were influenced primarily by Darwinian selection. The duplication time of the AP2/EREBP paralogous gene pairs in Z. mays L. ranged from approximately 9.364 to 100.935 Mya. The duplication time of the COX-3/COX-4 paralogous gene pair was approximately 5.217 Mya. The duplication time of the LTP paralogous gene pairs ranged from approximately 19.064 to 96.477 Mya. The major focus of research is to identify the genes that are responsible for drought stress tolerance to improve maize for drought stress tolerance. The results of the present study will improve the understanding of the functions of the AP2/EREBP, COX and LTP genes in response to drought stress.

Shotgun proteomics and in silico analysis of potential allergens in mature seeds and sprouts of purple winged bean

Winged bean (Psophocarpus tetragonolobus (L.) DC.) is a tropical legume crop found in Southeast Asian countries including Thailand. The mature seeds have high protein content and have been targeted as a protein source, and germination has been used to increase the value of nutrition. We used nanoLC/timsTOF Pro 2 combined with PEAKS® studio Xpro software to explore the proteomes of mature seeds and sprouts of purple winged bean. This study is the first report on the allergenic proteins in mature seeds and sprouts of purple winged bean. In silico analysis using Allergen Nomenclature search and the Allermatchtm program, 37 allergens were found. Conglutin beta 5 and late embryogenesis abundant protein (3 and D-29) were identified only in mature seeds, while 4 types of major pollen allergen Bet V, profilin and isoflavone reductase, were identified only in sprouts. Examples of allergens in both samples include basic 7S globulin, beta-conglycinin alpha/beta subunit (1,2), convicilin, defensin-like protein, 7 types of non-specific lipid-transfer protein, P24 oleosin isoform B. Since food allergy and airway allergy are found in sprouts, we used shotgun proteomics and in silico analysis for detecting the profiles of allergens. With regards to food safety, purple winged beans may also induce cross-reactivity in patients who are allergic to peanuts or other legumes. Therefore, identifying the proteomes and potential allergens in mature seeds and sprouts of purple winged beans could assist researchers in studying plant-based food safety.

COBRAPed cohort: Do sensitization patterns differentiate children with severe asthma from those with a milder disease?

It is unclear whether sensitization patterns differentiate children with severe recurrent wheeze (SRW)/severe asthma (SA) from those with non-severe recurrent wheeze (NSRW)/non-severe asthma (NSA). Our objective was to determine whether sensitization patterns can discriminate between children from the French COBRAPed cohort with NSRW/NSA and those with SRW/SA. IgE to 112 components (c-sIgE) (ImmunoCAP® ISAC) were analyzed in 125 preschools (3-6 years) and 170 school-age children (7-12 years). Supervised analyses and clustering methods were applied to identify patterns of sensitization among children with positive c-sIgE. We observed c-sIgE sensitization in 51% of preschool and 75% of school-age children. Sensitization to house dust mite (HDM) components was more frequent among NSRW than SRW (53% vs. 24%, p < .01). Sensitization to non-specific lipid transfer protein (nsLTP) components was more frequent among SA than NSA (16% vs. 4%, p < .01) and associated with an FEV1/FVC < -1.64 z-score. Among sensitized children, seven clusters with varying patterns were identified. The two broader clusters identified in each age group were characterized by "few sensitizations, mainly to HDM." One cluster (n = 4) with "multiple sensitizations, mainly to grass pollen, HDM, PR-10, and nsLTP" was associated with SA in school-age children. Although children with wheeze/asthma display frequent occurrences and high levels of sensitization, sensitization patterns did not provide strong signals to discriminate children with severe disease from those with milder disease. These results suggest that the severity of wheeze/asthma may depend on both IgE- and non-IgE-mediated mechanisms.

Lipid transfer protein StLTPa enhances potato disease resistance against different pathogens by binding and disturbing the integrity of pathogens plasma membrane.

Potato is the third most important food crop worldwide. Potato production suffers from severe diseases caused by multiple detrimental plant pathogens, and broad-spectrum disease resistance genes are rarely identified in potato. Here we identified the potato non-specific lipid transfer protein StLTPa, which enhances species none-specific disease resistance against various pathogens, such as the oomycete pathogen Phytophthora infestans, the fungal pathogens Botrytis cinerea and Verticillium dahliae, and the bacterial pathogens Pectobacterium carotovorum and Ralstonia solanacearum. The StLTPa overexpression potato lines do not show growth penalty. Furthermore, we provide evidence that StLTPa binds to lipids present in the plasma membrane (PM) of the hyphal cells of P. infestans, leading to an increased permeability of the PM. Adding of PI(3,5)P2 and PI(3)P could compete the binding of StLTPa to pathogen PM and reduce the inhibition effect of StLTPa. The lipid-binding activity of StLTPa is essential for its role in pathogen inhibition and promotion of potato disease resistance. We propose that StLTPa enhances potato broad-spectrum disease resistance by binding to, and thereby promoting the permeability of the PM of the cells of various pathogens. Overall, our discovery illustrates that increasing the expression of a single gene in potato enhances potato disease resistance against different pathogens without growth penalty.

Tamyb10-D1 restores red grain color and increases grain dormancy via suppressing expression of TaLTP2.128, non-specific lipid transfer protein in wheat

Grain dormancy of wheat is closely associated with grain color: red-grained lines show higher dormancy than white-grained lines. The production of red pigments is regulated by R-1, Tamyb10 gene. However, the relation between grain color and dormancy remains unknown. For this study, we generated transgenic lines which were introduced a DNA fragment containing Tamyb10-D1 gene and its a 2 kb promoter including the 5′ untranslated region into white-grained wheat. Transgenic lines showed red-grained and higher dormant traits. Contents of plant hormones and gene expression of embryos at 30 days after pollination were examined in a wild type and a transgenic line. No differences were observed in the contents of plant hormones, but several genes are differentially expressed between these lines. One differentially expressed gene, TaLTP2.128, is a member of non-specific lipid transfer proteins. It was expressed higher in white grains than in red grains. A putative amino acid sequence showed similarity to that of OsHyPRP5, which is identified as QTL controlling low-temperature germinability in rice. Expression of TaLTP2.128 was increased by grain imbibition. The increasing levels were higher not only in other white-grained lines, but also in non-dormant red-grained lines. TaLTP2.128 was expressed at a quite early stage of germination. These study findings indicate that Tamyb10 regulates dormancy release by the modification of TaLTP2.128 acting as trigger of germination.

Occupational hops allergy with nonspecific lipid transfer protein sensitization: Cross-reactivity with food nonspecific lipid transfer protein

Occupational hops allergy with nonspecific lipid transfer protein sensitization: Cross-reactivity with food nonspecific lipid transfer protein

English

Background: Sunflower seed is one of the most common edible seeds and its consumption is growing. Case reports of sunflower seed allergy have been described since the 1970s. However, there are few data on the prevalence and clinical manifestations of sunflower seed allergy. Objective: To improve understanding of sunflower seed allergy. Methods: We evaluated the clinical and immunological features of patients with sunflower seed allergy diagnosed in the Allergy Department of a tertiary hospital in Madrid over a 5-years period. Results: Forty-seven patients reported adverse reactions after ingestion of sunflower seed and had specific sensitization to sunflower seed determined by skin prick test (median 8 mm) or specific IgE (median 1.10 kUA/L). Most had an adult-onset reaction to sunflower seed preceded by a history of atopy and other food allergies, predominantly to peach, peanut and nuts. Clinical presentation of sunflower seed allergy ranged from mild to severe, with a high proportion of patients suffering severe reactions, often undertreated. A variability in the severity of symptoms was seen on repeated exposures to sunflower seed on a same patient. Levels of sunflower seed IgE were strongly correlated with levels of IgE to non-specific lipid transfer proteins, while no significant differences were found in the severity of the reactions according to sensitization to those proteins. Conclusion: Our findings reveal a variability of clinical presentations of sunflower seed allergy on repeated exposures and an underuse of epinephrine in anaphylaxis. We highlight the importance of strict avoidance of sunflower seed and accurate prescription and administration of epinephrine in allergic patients.

A Novel Non-Specific Lipid Transfer Protein Gene, CmnsLTP6.9, Enhanced Osmotic and Drought Tolerance by Regulating ROS Scavenging and Remodeling Lipid Profiles in Chinese Chestnut (Castanea mollissima Blume).

Chestnut (Castanea mollissima Blume) is an important economic tree owing to its tasty fruit and adaptability to environmental stresses, especially drought. Currently, there is limited information about non-specific lipid transfer protein (nsLTP) genes that respond to abiotic stress in chestnuts. Here, a chestnut nsLTP, named CmnsLTP6.9, was identified and analyzed. The results showed that the CmnsLTP6.9 protein localized in the extracellular matrix had two splicing variants (CmnsLTP6.9L and CmnsLTP6.9S). Compared with CmnsLTP6.9L, CmnsLTP6.9S had an 87 bp deletion in the 5'-terminal. Overexpression of CmnsLTP6.9L in Arabidopsis enhanced tolerance to osmotic and drought stress. Upon exposure to osmotic and drought treatment, CmnsLTP6.9L could increase reactive oxygen species (ROS)-scavenging enzyme activity, alleviating ROS damage. However, CmnsLTP6.9S-overexpressing lines showed no significant differences in phenotype, ROS content, and related enzyme activities compared with the wild type (WT) under osmotic and drought treatment. Moreover, lipid metabolism analysis confirmed that, unlike CmnsLTP6.9S, CmnsLTP6.9L mainly altered and upregulated many fatty acyls and glycerophospholipids, which implied that CmnsLTP6.9L and CmnsLTP6.9S played different roles in lipid transference in the chestnut. Taken together, we analyzed the functions of CmnsLTP6.9L and CmnsLTP6.9S, and demonstrated that CmnsLTP6.9L enhanced drought and osmotic stress tolerance through ROS scavenging and lipid metabolism.

Cyclophilins and gibberellin-regulated proteins in IgE-mediated allergic diseases

SummaryThe prevalence of immunoglobulin E (IgE)-mediated allergic diseases is currently experiencing an epidemic trend characterized by an increase in both the number of affected individuals and the proportion of patients with multiple sensitizations. The majority of these multiple sensitizations are attributed to IgE reactions to genuine allergenic proteins from unrelated species. However, there is a growing trend of patients becoming sensitized to highly cross-reactive molecules, such as profilins, polcalcins, lipocalins, serum albumins, tropomyosins, and non-specific lipid transfer proteins (nsLTPs). In addition, allergen families that were previously considered of minimal importance are now gaining recognition for their role in the pathogenesis of IgE-mediated allergic diseases. Consequently, these allergen families are increasingly being considered in the diagnostic process. In this review, we aim to provide a comprehensive summary of the biochemical and allergological information about two of these “new” allergen families: cyclophilins (Cyp) and gibberellin-regulated proteins (GRP).

Sensitivity, specificity and positive predictive value of ALEX2 multiplex examination in patients suffering from atopic dermatitis and reaction to tomatoes

ABSTRACT The aim of the study is to evaluate the clinical reactions to tomatoes in atopic dermatitis (AD) patients, and to analyse the results of specific IgE to allergens of tomatoes and to molecular components of pollen, seeds, fruits, vegetables and latex with the use of ALEX2 Allergy Xplorer test. According to our results, 15% of AD patients suffer from clinical reactions to tomatoes; we confirmed the low sensitivity and low positive predictive value of specific IgE to molecular component Sola l 6 and allergen extract Sola l, the specificity of this test is higher. The significantly higher sensitisation to other nonspecific lipid transfer proteins is observed in patients sensitised to tomato allergens. An elimination diet and an exposure test are recommended to detect a reaction to tomatoes. ALEX2 Allergy Xplorer test gives us a comprehensive picture of sensitisation and the possibility of evaluation of cross-reacting allergens.

IgE-mediated reactivity to non-specific lipid transfer protein (nsLTP): clinical implications and management ‒ consensus document of the Association of Italian Territorial and Hospital Allergists and Immunologists (AAIITO).

The primary cause of adult-onset food allergy in Mediterranean countries is IgE-mediated reactivity to non-specific Lipid Transfer Protein (nsLTP), with a prevalence of 9.5% in Italy. nsLTP is heat- and pepsin-stable due to its 3D structure, causing severe allergic reactions, even anaphylaxis. It's conserved across plants and a "panallergen" due to homologous forms in various vegetable foods. Found in Rosaceae fruits' skin, it's categorized into nsLTP1 (9 kDa) and nsLTP2 (7 kDa), representing 93% and 7% of the molecules described to date, respectively. Pru p 3 (nsLTP1) from peach is a primary sensitizer, binding more epitopes than other homologs. Cross-reactivity varies in sensitized patients, influenced by IgE levels. Clinical manifestations range from none to various symptoms. Managing patients sensitized to nsLTP without clinical allergy is a challenge. Sensitization hierarchy usually starts with peach, then expands through Prunoideae, Rosaceae, and other foods. Clinical symptoms don't always expand across LTPs. Patients can tolerate some nsLTP-containing foods and consuming them may maintain tolerance. The absence of guidelines led to the Associazione Allergologi Immunologi Italiani Territoriali e Ospedalieri (AAIITO) creating a consensus-based document. Strategies involve avoidance, self-injectable adrenaline, verification through in vivo and in vitro testing, considering cofactors, and peeling fruits. In localized reactions, abstinence is recommended if specific IgE is high. Concurrent pollinosis may complicates diagnosis, but may help management since symptoms are often less severe. Asymptomatic patients are advised to continue normal diets while considering cofactors and total IgE levels. Management strategies should be case-specific, based on expert Consensus Document.

Factors and co-factors influencing clinical manifestations in nsLTPs allergy: between the good and the bad.

Non-specific lipid transfer proteins (nsLTPs) are a family of plant pan-allergens that represent the primary cause of food allergies in the Mediterranean area, characterized by a wide range of clinical manifestations, ranging from the total absence of symptoms up to anaphylaxis. This wide variety of symptoms is related to the intrinsic capacity of nsLTPs to cause an allergic reaction in a specific subject, but also to the presence of co-factors exacerbating (i.e., exercise, NSAIDs, PPIs, alcohol, cannabis, prolonged fasting, menstruation, acute infections, sleep deprivation, chronic urticaria) or protecting from (i.e., co-sensitization to PR10, profilin or polcalcin) severe reactions. In this picture, recognizing some nsLTPs-related peculiarities (i.e., route, type and number of sensitizations, concentration of the allergen, cross-reactions) and eventual co-factors may help the allergist to define the risk profile of the single patient, in order to promote the appropriate management of the allergy from dietary advices up to the prescription of life-saving epinephrine autoinjector.

Identification and evolution of nsLTPs in the root nodule nitrogen fixation clade and molecular response of Frankia to AgLTP24

Non-specific lipid transfer proteins (nsLTPs) are antimicrobial peptides, involved in several plant biological processes including root nodule nitrogen fixation (RNF). Nodulating plants belonging to the RNF clade establish symbiosis with the nitrogen-fixing bacteria rhizobia (legumes symbiosis model) and Frankia (actinorhizal symbiosis model) leading to root nodule formation. nsLTPs are involved in processes active in early step of symbiosis and functional nodule in both models. In legumes, nsLTPs have been shown to regulate symbiont entry, promote root cortex infection, membrane biosynthesis, and improve symbiosis efficiency. More recently, a nsLTP, AgLTP24 has been described in the context of actinorhizal symbiosis between Alnus glutinosa and Frankia alni ACN14a. AgLTP24 is secreted at an early step of symbiosis on the deformed root hairs and targets the symbiont in the nitrogen-fixing vesicles in functional nodules. nsLTPs are involved in RNF, but their functions and evolutionary history are still largely unknown. Numerous putative nsLTPs were found up-regulated in functional nodules compared to non-infected roots in different lineages within the RNF clade. Here, results highlight that nodulating plants that are co-evolving with their nitrogen-fixing symbionts appear to have independently specialized nsLTPs for this interaction, suggesting a possible convergence of function, which opens perspectives to investigate nsLTPs functions in RNF.

Antifungal peptides from faba bean flour fermented by Levilactobacillus brevis AM7 improve the shelf-life of composite faba-wheat bread

Faba bean is an important crop from an ecological, nutritional, and economical perspective, hence its incorporation in plant-based products can play a key role in the sustainability of the whole food system. In this study, the antifungal properties of faba bean flour were investigated employing three bioprocessing treatments including hydrolysis with a commercial proteolytic enzyme preparation, and fermentation with selected or commercial mixed sourdough starters. Faba bean flour fermented by Levilactobacillus brevis AM7 showed the broadest inhibitory spectrum against the fungal species tested and antifungal activity ranging from 25 to 73 %. Bioactive proteins and peptides were found to be responsible for the antifungal activity. Polypeptides sequences corresponding to defensin-like and non-specific lipid-transfer proteins and seven antifungal peptides having 11–22 amino acid residues and mass from 1240.7 to 2624.2 Da, were identified via Liquid Chromatography-Electrospray Ionisation-Mass Spectra/Mass Spectra (nano-LC-ESI-MS/MS). To assess the antifungal effect in food processing conditions, fermented faba bean was used as ingredient in breadmaking at a substitution level of 30 % of the dough weight. Wheat composite-bread containing faba bean sourdough, which could be claimed as “source of proteins”, according to the current EC Regulation, delayed the molds contamination, lasting up to 10 days before mold appearance, and behaving similarly to wheat bread containing calcium propionate.

Proteomics-Based Approach for Detailing the Allergenic Profile of Cannabis Chemotypes.

Allergic sensitization to cannabis is an emerging public health concern and is difficult to clinically establish owing to lack of standardized diagnostic approaches. Attempts to develop diagnostic tools were largely hampered by the Schedule I restrictions on cannabis, which limited accessibility for research. Recently, however, hemp was removed from the classified list, and increased accessibility to hemp allows for the evaluation of its practical clinical value for allergy diagnosis. We hypothesized that the proteomic profile is preserved across different cannabis chemotypes and that hemp would be an ideal source of plant material for clinical testing. Using a proteomics-based approach, we examined whether distinct varieties of cannabis plant contain relevant allergens of cannabis. Cannabis extracts were generated from high tetrahydrocannabinol variety (Mx), high cannabidiol variety (V1-19) and mixed profile variety (B5) using a Plant Total Protein Extraction Kit. Hemp extracts were generated using other standardized methods. Protein samples were subjected to nanoscale tandem mass spectrometry. Acquired peptides sequences were examined against the Cannabis sativa database to establish protein identity. Non-specific lipid transfer protein (Can s 3) level was measured using a recently developed ELISA 2.0 assay. Proteomic analysis identified 49 distinct potential allergens in protein extracts from all chemotypes. Most importantly, clinically relevant and validated allergens, such as profilin (Can s 2), Can s 3 and Bet v 1-domain-containing protein 10 (Can s 5), were identified in all chemotypes at label-free quantification (LFP) intensities > 106. However, the oxygen evolving enhancer protein 2 (Can s 4) was not detected in any of the protein samples. Similarly, Can s 2, Can s 3 and Can s 5 peptides were also detected in hemp protein extracts. The validation of these findings using the ELISA 2.0 assay indicated that hemp extract contains 30-37 ng of Can s 3 allergen per µg of total protein. Our proteomic studies indicate that relevant cannabis allergens are consistently expressed across distinct cannabis chemotypes. Further, hemp may serve as an ideal practical substitute for clinical testing, since it expresses most allergens relevant to cannabis sensitization, including the validated major allergen Can s 3.

Potato Non-Specific Lipid Transfer Protein StnsLTPI.33 Is Associated with the Production of Reactive Oxygen Species, Plant Growth, and Susceptibility to Alternaria solani.

Plant non-specific lipid transfer proteins (nsLTPs) are small proteins capable of transferring phospholipids between membranes and binding non-specifically fatty acids in vitro. They constitute large gene families in plants, e.g., 83 in potato (Solanum tuberosum). Despite their recognition decades ago, very few have been functionally characterized. Here, we set out to better understand the function of one of the potato members, StnsLTPI.33. Using quantitative polymerase chain reaction, we show that StnsLTPI.33 is expressed throughout the potato plant, but at relatively higher levels in roots and leaves compared to petals, anthers, and the ovary. We also show that ectopically-expressed StnsLTPI.33 fused to green fluorescent protein colocalized with an apoplastic marker in Nicotiana benthamiana leaves, indicating that StnsLTPI.33 is targeted to the apoplast. Constitutive overexpression of the StnsLTPI.33 gene in potato led to increased levels of superoxide anions and reduced plant growth, particularly under salt stress conditions, and enhanced susceptibility to Alternaria solani. In addition, StnsLTPI.33-overexpressing plants had a depleted leaf pool of pipecolic acid, threonic acid, and glycine, while they accumulated putrescine. To our knowledge, this is the first report of an nsLTP that is associated with enhanced susceptibility to a pathogen in potato.