UNASSIGNED: Background. Gibberellin Regulated Proteins (GRPs) are small glycoproteins that induce allergy to various types of fruit. This study aimed to evaluate co-sensitization to cypress pollen and other molecules responsible for fruit allergy, such as nsLTP (Pru p 3), PR-10 (Bet v1), and Profilin (Bet v2). Methods. Sixty subjects sensitized to peach GRP (Pru p 7) were consecutively recruited from four Italian centers: 28 males and 32 females (mean age 37.9 years; range 11-79). Specific IgE for Pru p 7, Pru p 3, Bet v 1, Bet v 2, cypress pollen extract (Cup s), and Cup a 1 were determined in all subjects. Results. Sensitization rates to Cup s, Cup a 1, Pru p 3, Bet v 1, and Bet v 2 in the entire studied population were 90.0%, 83.3%, 45.8%, 40.0%, and 30.0%, respectively. In subjects residing in Northern Italy, the respective sensitization rates were 96.4%, 80.0%, 50.0%, 73.3%, and 40.0%, while in those residing in Southern Italy, they were 83.3%, 86.7%, 40.0%, 6.7%, and 20.0%. The only significant difference was observed for PR-10 (p less than 0.0001) Co-sensitization to PR-10 was found to be associated with a reduced risk of anaphylaxis (OR: 0.125). Allergic reactions were most commonly triggered by peach (26/40), followed by orange (12/40), with other foods being less frequently implicated. Conclusions. This study confirms a high association between sensitization to Pru p 7 and cypress pollen and highlights a high percentage of co-sensitization to nsLTP, PR-10, and profilin. PR-10 emerged as a protective factor against anaphylaxis.

Pollen from common ragweed is an important allergen source worldwide and especially in western and southern Romania. More than 100 million patients suffer from symptoms of respiratory allergy (e.g., rhinitis, asthma) to ragweed pollen. Among the eleven characterized allergens, Amb a 6 is a non-specific lipid transfer protein (nsLTP). nsLTPs are structurally stable proteins in pollen and food from different unrelated plants capable of inducing severe reactions. The goal of this study was to produce Amb a 6 as a recombinant and structurally folded protein (rAmb a 6) and to characterize its physicochemical and immunological features. rAmb a 6 was expressed in Spodoptera frugiperda Sf9 cells as a secreted protein and characterized by mass spectrometry and circular dichroism (CD) spectroscopy regarding molecular mass and fold, respectively. The IgE-binding frequency towards the purified protein was evaluated using sera from 150 clinically well-characterized ragweed-allergic patients. The allergenic activities of rAmb a 6 and the nsLTP from the weed Parietaria judaica (Par j 2) were evaluated in basophil activation assays. rAmb a 6-specific IgE reactivity was associated with clinical features. Pure rAmb a 6 was obtained by insect cell expression. Its deduced molecular weight corresponded to that determined by mass spectrometry (i.e., 10,963 Da). rAmb a 6 formed oligomers as determined by SDS-PAGE under non-reducing conditions. According to multiple sequence comparisons, Amb a 6 was a distinct nsLTP with less than 40% sequence identity to currently known plant nsLTP allergens, except for nsLTP from Helianthus (i.e., 52%). rAmb a 6 is an important ragweed allergen recognized by 30% of ragweed pollen allergic patients. For certain patients, rAmb a 6-specific IgE levels were higher than those specific for the major ragweed allergen Amb a 1 and analysis also showed a higher allergenic activity in the basophil activation test. rAmb a 6-positive patients suffered mainly from respiratory symptoms. The assumption that Amb a 6 is a source-specific ragweed allergen is supported by the finding that none of the patients showing rAmb a 6-induced basophil activation reacted with Par j 2 and only one rAmb a 6-sensitized patient had a history of plant food allergy. Immunization of rabbits with rAmb a 6 induced IgG antibodies which strongly inhibited IgE binding to rAmb a 6. Our results demonstrate that Amb a 6 is an important source-specific ragweed pollen allergen that should be considered for diagnosis and allergen-specific immunotherapy of ragweed pollen allergy.

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A significant fraction of allergens bind small molecular ligands, and many of these compounds are classified as lipids. However, in most cases, we do not know the role that is played by the ligands in the allergic sensitization or allergic effector phases.
More effort is dedicated toward identification of allergens\' ligands. This resulted in identification of some lipidic compounds that can play active immunomodulatory roles or impact allergens\' molecular and allergic properties. Four allergen families (lipocalins, NPC2, nsLTP, and PR-10) are among the best characterized in terms of their ligand-binding properties. Allergens from these four families are able to bind many chemically diverse molecules. These molecules can directly interact with human immune system and/or affect conformation and stability of allergens. While there is more data on the allergens and their small molecular ligands, we are just starting to understand their role in allergy.

Plant non-specific lipid transfer proteins (nsLTPs) are small basic proteins that play a significant regulatory role in a wide range of physiological processes. To date, no genome-wide survey and expression analysis of this gene family in sugarcane has been performed. In this study we identified the nsLTP gene family in Saccharum spontaneum and carried out expression profiling of nsLTPs in two sugarcane cultivars (Saccharum spp.) that have different resistance to leaf scald caused by Xanthomonas albilineans (Xa) infection. The effect of stress related to exogenous salicylic acid (SA) treatment was also examined. At a genome-wide level, S. spontaneum AP85-441 had 71 SsnsLTP genes including 66 alleles. Tandem (9 gene pairs) and segmental (36 gene pairs) duplication events contributed to SsnsLTP gene family expansion. Five SsnsLTP proteins were predicted to interact with five other proteins. Expression of ShnsLTPI.8/10/Gb.1 genes was significantly upregulated in LCP85-384 (resistant cultivar), but downregulated in ROC20 (susceptible cultivar), suggesting that these genes play a positive regulatory role in response of sugarcane to Xa infection. Conversely, ShnsLTPGa.4/Ge.3 appears to act as a negative regulator in response Xa infection. The majority (16/17) of tested genes were positively induced in LCP85-384 72 h after SA treatment. In both cultivars, but particularly in LCP85-384, ShnsLTPIV.3/VIII.1 genes were upregulated at all time-points, suggesting that the two genes might act as positive regulators under SA stress. Meanwhile, both cultivars showed downregulated ShnsLTPGb.1 gene expression, indicating its potential negative role in SA treatment responses. Notably, the ShnsLTPGb.1 gene had contrasting effects, with positive regulation of gene expression in response to Xa infection and negative regulation induced by SA stress. Together, our results provide valuable information for elucidating the function of ShnsLTP family members under two stressors and identified novel gene sources for development of sugarcane that are tolerant of environmental stimuli.

Non-specific lipid transfer proteins (nsLTPs) are small cysteine-rich basic proteins which play essential roles in plant growth, development and abiotic/biotic stress response. However, there is limited information about the nsLTP gene (BnLTP) family in rapeseed (Brassica napus). In this study, 283 BnLTP genes were identified in rapeseed, which were distributed randomly in 19 chromosomes of rapeseed. Phylogenetic analysis showed that BnLTP proteins were divided into seven groups. Exon/intron structure and MEME motifs both remained highly conserved in each BnLTP group. Segmental duplication and hybridization of rapeseed\'s two sub-genomes mainly contributed to the expansion of the BnLTP gene family. Various potential cis-elements that respond to plant growth, development, biotic/abiotic stresses, and phytohormone signals existed in BnLTP gene promoters. Transcriptome analysis showed that BnLTP genes were expressed in various tissues/organs with different levels and were also involved in the response to heat, drought, NaCl, cold, IAA and ABA stresses, as well as the treatment of fungal pathogens (Sclerotinia sclerotiorum and Leptosphaeria maculans). The qRT-PCR assay validated the results of RNA-seq expression analysis of two top Sclerotinia-responsive BnLTP genes, BnLTP129 and BnLTP161. Moreover, batches of BnLTPs might be regulated by BnTT1 and BnbZIP67 to play roles in the development, metabolism or adaptability of the seed coat and embryo in rapeseed. This work provides an important basis for further functional study of the BnLTP genes in rapeseed quality improvement and stress resistance.

BACKGROUND: Trachyspermum ammi, commonly known as Ajwain, is a member of the Apiaceae family. It is a therapeutic herbal spice with diverse pharmacological properties, used in traditional medicine for various ailments. However, all previous studies were conducted using small molecule extracts, leaving the protein\'s bioactivity undiscovered.
OBJECTIVE: The current study aimed to demonstrate the cytotoxic activity of Ajwain non-specific lipid transfer protein (nsLTP1) in normal breast (MCF10A), breast cancer (MCF-7), and pancreatic cancer (AsPC-1) cell lines. Also, to evaluate its structural stability in human serum as well as at high temperature conditions.
METHODS: The cytotoxic activity of Ajwain nsLTP1 was evaluated in MCF-7 and AsPC-1 cell lines using MTT assay. Annexin V-FITC and PI staining were used to detect the early apoptotic and late apoptotic cells. The role of nsLTP1 in inducing apoptosis was further studied by quantifying Bcl-2, Bax, Caspase-3, Survivin, EGFR, and VEGF genes expression using RT-PCR. CD spectroscopy analyzed the nsLTP1 conformational changes after thermal treatment for structure stability determination. The RP-HPLC was used to analyze the nsLTP1 degradation rate in human serum at different time intervals incubated at 37 °C.
RESULTS: Ajwain nsLTP1 showed a potent cytotoxic effect in MCF-7 and AsPC-1. The IC50 value obtained in MCF-7 was 8.21 μM, while for AsPC-1 4.17 μM. The effect of nsLTP1 on stimulating apoptosis revealed that the proportions of apoptotic cells in both cell lines were relatively increased depending on the concentration. The apoptotic cells percentage at 20 μM was in MCF-7 71% (***P < 0.001) and AsPC-1 88% (***P < 0.001). These results indicate that nsLTP1 might efficaciously induce apoptosis in multiple types of cancerous cells. Genes expression in MCF-7 and AsPC-1 showed significant upregulation in Bax and Caspase-3 and downregulation in Bcl-2, Survivin, EGFR, and VEGF protein. The CD analysis of nsLTP1 showed a significant thermostable property. In serum, nsLTP1 showed a slow degradation rate, indicating high stability with a half-life of ~ 8.4 h.
CONCLUSIONS: Our results revealed the potential anticancer activity of Ajwain nsLTP1 and its mechanism in inducing apoptosis. It further exhibited thermostable properties at high temperatures and in human serum, which suggested this protein as a promising anticancer agent.

Pectin, a dietary fiber, is a polysaccharide that is widely used in food industry as a gelling agent. In addition, prebiotic and beneficial immunomodulatory effects of pectin have been demonstrated, leading to increased importance as food supplement. However, as cases of anaphylactic reactions after consumption of pectin-supplemented foods have been reported, the present study aims to evaluate the allergy risk of pectin. This is of particular importance since most of the pectin used in the food industry is extracted from citrus or apple pomace. Both contain several allergens such as non-specific lipid transfer proteins (nsLTPs), known to induce severe allergic reactions, which could impair the use of pectins in nsLTP allergic patients. Therefore, the present study for the first time was performed to analyze residual nsLTP content in two commercial pectins using different detection methods. Results showed the analytical sensitivity was diminished by the pectin structure. Finally, spiking of pectin with allergenic peach nsLTP Pru p 3 led to the conclusion that the potential residual allergen content in both pectins is below the threshold to induce anaphylactic reactions in nsLTP-allergic patients. This data suggests that consumption of the investigated commercial pectin products provides no risk for inducing severe reactions in nsLTP-allergic patients.

BACKGROUND: Brassica napus is an important agricultural species, improving stress resistance was one of the main breeding goals at present. Non-specific lipid transfer proteins (nsLTPs) are small, basic proteins which are involved in some biotic or abiotic stress responses. B. napus is susceptible to a variety of fungal diseases, so identify the BnLTPs and their expression in disease responses is very important. The common reference genome of B. napus does not contain all B. napus genes because of gene presence/absence variations between individuals. Therefore, it was necessary to search for candidate BnLTP genes in the B. napus pangenome.
RESULTS: In the present study, the BnLTP genes were identified throughout the pangenome, and different BnLTP genes were presented among varieties. Totally, 246 BnLTP genes were identified and could be divided into five types (1, 2, C, D, and G). The classification, phylogenetic reconstruction, chromosome distribution, functional annotation, and gene expression were analyzed. We also identified potential cis-elements that respond to biotic and abiotic stresses in the 2 kb upstream regions of all BnLTP genes. RNA sequencing analysis showed that the BnLTP genes were involved in the response to Sclerotinia sclerotiorum infection. We identified 32 BnLTPs linked to blackleg resistance quantitative trait locus (QTL).
CONCLUSIONS: The identification and analysis of LTP genes in the B. napus pangenome could help to elucidate the function of BnLTP family members and provide new information for future molecular breeding in B. napus.

Food allergy to sunflower seed (SFS) protein is not frequent and only non-specific lipid transfert protein (nsLTP) Hel a 3 is officially recognized as a food allergen. Out of the eleven seed storage 2S-albumins (SESA) detected in SFS, only SFA-8 allergenicity has been investigated so far. The study aimed then to evaluate SFS protein allergenicity and particularly, to compare the sensitization potency of SESA in a mouse model.
The most abundant SESA and nsLTP were isolated from SFS through a combination of chromatographic methods. Purified proteins were then used to measure specific IgG1 and IgE responses in BALB/c mice orally sensitized to different SFS protein isolates. The study, thus, confirmed the allergenicity of SFA-8 and Hel a 3 but mice were also highly sensitized to other SESA such as SESA2-1 or SESA20-2. Furthermore, competitive inhibition of IgE-binding revealed that SFA-8 IgE-reactivity was due to cross-reactivity with other SESA. 11S-globulins were weakly immunogenic and were rapidly degraded in an in vitro model of gastroduodenal digestion. In contrast, Hel a 3, SESA2-1 and SFA-8 were more resistant to proteolysis and gastroduodenal digestion did not affect their IgE-reactivity.
SESA2-1 or SESA20-2 were more potent allergens than SFA-8 in this mouse model. Allergenicity of SESA must be now confirmed in SFS-allergic patients.