Editing of an Alpha-Kafirin Gene Family Increases, Digestibility and Protein Quality in Sorghum.

Kafirins are the major storage proteins in sorghum (Sorghum bicolor) grains and form protein bodies with poor digestibility. Since kafirins are devoid of the essential amino acid lysine, they also impart poor protein quality to the kernel. The α-kafirins, which make up most of the total kafirins, are largely encoded by the k1C family of highly similar genes. We used a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to target the k1C genes to create variants with reduced kafirin levels and improved protein quality and digestibility. A single guide RNA was designed to introduce mutations in a conserved region encoding the endoplasmic reticulum signal peptide of α-kafirins. Sequencing of kafirin PCR products revealed extensive edits in 25 of 26 events in one or multiple k1C family members. T1 and T2 seeds showed reduced α-kafirin levels, and selected T2 events showed significantly increased grain protein digestibility and lysine content. Thus, a single consensus single guide RNA carrying target sequence mismatches is sufficient for extensive editing of all k1C genes. The resulting quality improvements can be deployed rapidly for breeding and the generation of transgene-free, improved cultivars of sorghum, a major crop worldwide.

Pea Ferritin Stability under Gastric pH Conditions Determines the Mechanism of Iron Uptake in Caco-2 Cells.

Background: Iron deficiency is an enduring global health problem that requires new remedial approaches. Iron absorption from soybean-derived ferritin, an ∼550-kDa iron storage protein, is comparable to bioavailable ferrous sulfate (FeSO4). However, the absorption of ferritin is reported to involve an endocytic mechanism, independent of divalent metal ion transporter 1 (DMT-1), the transporter for nonheme iron. Objective: Our overall aim was to examine the potential of purified ferritin from peas (Pisum sativum) as a food supplement by measuring its stability under gastric pH treatment and the mechanisms of iron uptake into Caco-2 cells. Methods: Caco-2 cells were treated with native or gastric pH-treated pea ferritin in combination with dietary modulators of nonheme iron uptake, small interfering RNA targeting DMT-1, or chemical inhibitors of endocytosis. Cellular ferritin formation, a surrogate measure of iron uptake, and internalization of pea ferritin with the use of specific antibodies were measured. The production of reactive oxygen species (ROS) in response to equimolar concentrations of native pea ferritin and FeSO4 was also compared. Results: Pea ferritin exposed to gastric pH treatment was degraded, and the released iron was transported into Caco-2 cells by DMT-1. Inhibitors of DMT-1 and nonheme iron absorption reduced iron uptake by 26-40%. Conversely, in the absence of gastric pH treatment, the iron uptake of native pea ferritin was unaffected by inhibitors of nonheme iron absorption, and the protein was observed to be internalized in Caco-2 cells. Chlorpromazine (clathrin-mediated endocytosis inhibitor) reduced the native pea ferritin content within cells by ∼30%, which confirmed that the native pea ferritin was transported into cells via a clathrin-mediated endocytic pathway. In addition, 60% less ROS production resulted from native pea ferritin in comparison to FeSO4. Conclusion: With consideration that nonheme dietary inhibitors display no effect on iron uptake and the low oxidative potential relative to FeSO4, intact pea ferritin appears to be a promising iron supplement.

Effect of iron status in rats on the absorption of metal ions from plant ferritin.

An isolate of lead-ferritin obtained from soybean seeds sprouted in 25 mM of PbNO3 was introduced into the diet of both iron-deficient and iron non-deficient male rats. After a 21-day administration period, statistical differences in the lead accumulation in the femurs of the rats were noted. Iron-deficient rats accumulated more than four times the amount of lead in their bones than rats without iron-deficiency. No further decrease was observed in haemoglobin concentrations in the groups of animals fed with lead isolates, either iron-deficient or iron non-deficient. Also, no differences in the mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV) were observed at the end of the experiment in the group of iron non-deficient rats fed with lead-ferritin isolate compared to the control group of iron non-deficient rats. In the iron-deficient group fed with lead-ferritin isolate, a small increase in haemoglobin concentrations, MCH, MCV and mean corpuscular haemoglobin concentrations (MCHC) was recorded. The results presented in this paper confirm that lead from the tested preparation-lead ferritin isolate-was better absorbed by those rats with induced iron deficiency anaemia. Additionally, we may also suspect based on the obtained results that absorption of ferritin-iron depends on iron status in the body.

Evaluation of riproximin binding properties reveals a novel mechanism for cellular targeting.

Riproximin is a cytotoxic type II ribosome-inactivating protein showing high selectivity for tumor cell lines. Its binding to cell surface glycans is crucial for subsequent internalization and cytotoxicity. In this paper, we describe a unique mechanism of interaction and discuss its implications for the cellular targeting and cytotoxicity of riproximin. On a carbohydrate microarray, riproximin specifically bound to two types of asialo-glycans, namely to bi- and triantennary complex N-glycan structures (NA2/NA3) and to repetitive N-acetyl-D-galactosamine (GalNAc), the so-called clustered Tn antigen, a cancer-specific O-glycan on mucins. Two glycoproteins showing high riproximin binding, the NA3-presenting asialofetuin and the clustered Tn-rich asialo-bovine submaxillary mucin, were subsequently chosen as model glycoproteins to mimic the binding interactions of riproximin with the two types of glycans. ELISA analyses were used to relate the two binding specificities of riproximin to its two sugar binding sites. The ability of riproximin to cross-link the two model proteins revealed that binding of the two types of glycoconjugates occurs within different binding sites. The biological implications of these binding properties were analyzed in cellular assays. The cytotoxicity of riproximin was found to depend on its specific and concomitant interaction with the two glycoconjugates as well as on dynamic avidity effects typical for lectins binding to multivalent glycoproteins. The presence of definite, cancer-related structures on the cells to be targeted determines the therapeutic potency of riproximin. Due to its cross-linking ability, riproximin is expected to show a high degree of specificity for cells exposing both NA2/NA3 and clustered Tn structures.

Expression of curcin-transferrin receptor binding peptide fusion protein and its anti-tumor activity.

Curcin can inhibit the proliferation of tumor cells and promote tumor cell apoptosis, but the cytotoxicity of curcin is not selective for tumors or normal cells. In order to enhance the targeting of the anti-tumor ability of curcin, a transferrin receptor (TfR) binding peptide, TfRBP9, was fused with curcin. The curcin-TfRBP9 gene was cloned into pQE-30 and the recombinant vector pQE-30-curcin-TfRBP9 was established. Then the recombinant vector pQE-30-curcin-TfRBP9 was transferred into Escherichia coli M15. After being induced by 0.5mM IPTG for 6h at 37°C, the expressed quantity of the recombinant protein was about 30% of the total protein. Recombinant curcin-TfRBP9 was expressed in the form of an inclusion body. After dissolution, purification and renaturation, the purity of the recombinant curcin-TfRBP9 reached 95%. Immunofluorescence analysis showed that the TfRBP9 significantly enhanced the ability of the curcin binding to HepG2, and was enriched in the cytoplasm. The curcin-TfRBP9 fusion protein had significant proliferation inhibition effects on the HepG2 cells that over-expressed transferrin receptors, had lower inhibitory effects on the SKBR-3 cells that expressed low transferrin receptors, and had the lowest inhibitory effects on the LO-2 cells that were normal human liver cells. Compared with curcin, the curcin-TfRBP9 induced higher apoptosis rates in the HepG2 cells.

Pea protein provides a promising matrix for microencapsulating iron.

Worldwide, the most prevalent nutritional deficiency is iron. The strategies for iron supplementation often fail due to poor adherence to supplementation methods contributed to unpleasant sensory characteristics. An alternative is the use of microencapsulated nutrients for home fortification in order to mask undesirable tastes and to allow its release in strategic sites of the gastrointestinal tract. Toward this end, pea protein concentrate was tested as a natural, edible and alternative material and the spray-drying technique was utilized for the preparation of microparticles containing ferrous sulfate. Their physical and chemical characteristics were evaluated. The microparticles had a spherical shape and grooves with an average size ranging between 2 and 3 µm. Analysis by in vitro assays tested the release of iron in simulated salivary and gastric fluids and its intestinal absorption in Caco-2 cells. No dissolution of iron occurred in the salivary medium whereas the sensory analysis showed good acceptance of a product which incorporated 5.5 mg of iron per 100 g portion of food. Thus, the effectiveness of microencapsulation was demonstrated by utilizing a plant protein as an encapsulating matrix for the controlled release of iron and capable of preserving the bioaccessibility of ferrous sulfate.

Purification and characterization of Mn-peroxidase from Musa paradisiaca (banana) stem juice.

Mn-peroxidase (MnP), a biotechnologically important enzyme was purified for the first time from a plant source Musa paradisiaca (banana) stem, which is an agro-waste easily available after harvest of banana fruits. MnP was earlier purified only from the fungal sources. The enzyme was purified from stem juice by ultrafiltration and anion-exchange column chromatography on diethylamino ethylcellulose with 8-fold purification and purification yield of 65%. The enzyme gave a single protein band in SDS-PAGE corresponding to molecular mass 43 kDa. The Native-PAGE of the enzyme also gave a single protein band, confirming the purity of the enzyme. The UV/VIS spectrum of the purified enzyme differed from the other heme peroxidases, as the Soret band was shifted towards lower wavelength and the enzyme had an intense absorption band around 250 nm. The K(m) values using MnSO4 and H2O2 as the substrates of the purified enzyme were 21.0 and 9.5 microM, respectively. The calculated k(cat) value of the purified enzyme using Mn(II) as the substrate in 50 mM lactate buffer (pH 4.5) at 25 degrees C was 6.7s(-1), giving a k(cat)/K(m) value of 0.32 microM(-1)s(-1). The k(cat) value for the MnP-catalyzed reaction was found to be dependent of the Mn(III) chelator molecules malonate, lactate and oxalate, indicating that the enzyme oxidized chelated Mn(II) to Mn(III). The pH and temperature optima of the enzyme were 4.5 and 25 degrees C, respectively. The enzyme in combination with H2O2 liberated bromine and iodine in presence of KBr and KI respectively. All these enzymatic characteristics were similar to those of fungal MnP. The enzyme has the potential as a green brominating and iodinating agent in combination with KBr/KI and H2O2.

Impact of soy lecithin, zinc oxide, and methylsulfonylmethane, as excipient ingredients, on the bioaccessibility and intestinal transport of branched-chain amino acids from animal and plant protein mixtures.

To maximize the biological activity of branched-chain amino acids (BCAAs), it is necessary to find a new excipient agent to increase the bioavailability of BCAAs in protein mixtures. The aim of the current study was to investigate the effects of soy lecithin (SLC), zinc oxide (ZnO), and methylsulfonylmethane (MSM) on the bioaccessibility and intestinal transport of BCAAs from animal and plant protein mixtures (PMs) via an in vitro digestion model with human intestinal epithelial (Caco-2) cells. The bioaccessibility of total BCAAs in PMs considerably increased by 107.51 ± 1.50% with the addition of SLC, and the combined effects of SLC, ZnO, and MSM on enhancing the bioaccessibility of total BCAAs was observed (107.14 ± 0.18%). Interestingly, SLC showed a major role in binding bile acid, showing 65.78 ± 1.66% of binding capacity. Intestinal transport of BCAAs was measured to be at 100.48, 110.86, and 130.29 µg mL-1 for leucine, isoleucine, and valine, respectively, in PMs with SLC + ZnO + MSM, and it eventually amplified the amount of the total transported BCAAs (341.63 ± 6.34 µg mL-1), which was about 8.72 times higher than that of PM only. The cellular integrity of digesta-treated Caco-2 cells tended to decrease according to the incubation time, but it was recovered in the treatment of PM + SLC + ZnO + MSM, and nearly reached the control levels with 92.82 ± 0.53%. Results from the current study suggest that the co-consumption of proteins equally consisting of plant and animal sources with SLC, ZnO, and MSM could improve the bioavailability of total BCAAs, resulting in the improvement of health benefits.

Trans-Epithelial Transport, Metabolism, and Biological Activity Assessment of the Multi-Target Lupin Peptide LILPKHSDAD (P5) and Its Metabolite LPKHSDAD (P5-Met).

P5 (LILPKHSDAD) is a hypocholesterolemic peptide from lupin protein with a multi-target activity, since it inhibits both 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) and proprotein convertase subtilisin/kexin type-9 (PCSK9). This work shows that, during epithelial transport experiments, the metabolic transformation mediated by intestinal peptidases produces two main detected peptides, ILPKHSDAD (P5-frag) and LPKHSDAD (P5-met), and that both P5 and P5-met are linearly absorbed by differentiated human intestinal Caco-2 cells. Extensive comparative structural, biochemical, and cellular characterizations of P5-met and the parent peptide P5 demonstrate that both peptides have unique characteristics and share the same mechanisms of action. In fact, they exert an intrinsically multi-target behavior being able to regulate cholesterol metabolism by modulating different pathways. The results of this study also highlight the dynamic nature of bioactive peptides that may be modulated by the biological systems they get in contact with.

True ileal amino acid digestibility and digestible indispensable amino acid scores (DIAASs) of plant-based protein foods.

Plant-based protein foods are increasingly common, but data on their nutritional protein quality are scarce. This study evaluated it for seitan (wheat-based food), tofu (soya-based food), soya milk, and a pea emulsion. The true ileal digestibility (TID) of their amino acids was determined in minipigs, to calculate the digestible indispensable amino acid score (DIAAS). The TID of the proteins was high and not significantly different between the foods tested: 97% for seitan, 95% for tofu, 92% for soya milk and 94% for pea emulsion. There were only minor differences in individual amino acid TIDs. DIAAS ranking was thus essentially driven by the amino acid composition of the food: soya-based food > pea emulsion > seitan. Nevertheless, the lower TID of sulphur-containing amino acids in tofu than in soya milk induced a significant decrease in DIAAS (from 117% to 97%), highlighting the importance of the matrix effect on nutritional protein quality.

Characterization of the key region and putative phosphorylation sites of EcaICE1 in its molecular interaction with the EcaHOS1 protein in Eucalyptus camaldulensis.

Inducer of CBF expression 1 (ICE1), a MYC-like bHLH transcriptional activator, plays an important role in plants under cold stress. The ubiquitination-proteasome pathway mediated by high expression of osmotically responsive gene1 (HOS1) can effectively induce the degradation of ICE1 and decrease the expression of CBFs and their downstream genes under cold stress response in Arabidopsis, but knowledge of ubiquitination regulation of ICE1 by HOS1 is still limited in woody plants. In this study, a E3 ubiquitin ligase gene EcaHOS1 were amplified from Eucalyptus camaldulensis and the protein interactions between EcaICE1 and EcaHOS1 were analysed. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assay results showed that EcaICE1 can interact with the EcaHOS1 protein in the nucleus and, further, the Y2H assay demonstrated that the 126-185 amino acid region at the N-terminus of the EcaICE1 protein was indispensable for its interaction with EcaHOS1 protein. Moreover, we found that the amino acids at positions 145, 158 and 184 within the key interaction region were the putative phosphorylation sites of EcaICE1, based on bioinformatics analysis, and only the substitution of serine (Ser) 158 by alanine (Ala) blocked the protein-protein interactions between EcaICE1 and EcaHOS1 based on Y2H and ß-galactosidase activity assays using site-directed mutagenesis. We identified Ser 158 of EcaICE1 as the key putative phosphorylation site for its interaction with the EcaHOS1 protein.

In vitro gastrointestinal digestion and simulated colonic fermentation of pistachio nuts determine the bioaccessibility and biosynthesis of chronobiotics.

Chronodisruption leads to obesity and other metabolic disorders that can be alleviated by food-derived potential chronobiotics, such as phytomelatonin (PMT), phenolic compounds (PCs) and dietary fiber rich pistachios. Pistachios with (PN + SC) or without (PN) the seed coat were investigated for their in vitro chronobiotic potential since they are one of the main reported PMT sources. Consequently we evaluated the bioaccessibility, permeability, and biosynthesis of pistachio chronobiotics, particularly PMT, during gastrointestinal and colonic fermentation. The maximum in vitro bioaccessibility and apparent permeability (efflux-prone) of PCs, flavonoids and PMT were sample-specific [∼1.3% (both), 27 and 3.4% (PN + SC)], but additional amounts (flavonoids > PCs > PMT) were released under simulated colonic conditions. Short-chain fatty acids (SCFAs; 38 mM; >50% butyrate, PN + SC > PN) and some metabolites (e.g., indole, benzaldehyde, phenolic acids, and aliphatic/aromatic hydrocarbons) were detected depending on the sample. The predominant pistachio butyrate production during in vitro colonic fermentation can improve chronodisruption and benefit obese individuals. Pistachio's digestion increases the bioaccessibility and intestinal permeability of potential chronobiotics (PMT and PCs) and the biosynthesis of colonic metabolites (SCFAs, among others) also with chronobiotic potential.

Digestion and Transport across the Intestinal Epithelium Affects the Allergenicity of Ara h 1 and 3 but Not of Ara h 2 and 6.

SCOPE: No accepted and validated methods are currently available which can accurately predict protein allergenicity. In this study, the role of digestion and transport on protein allergenicity is investigated. METHODS AND RESULTS: Peanut allergens (Ara h 1, 2, 3, and 6) and a milk allergen (ß-lactoglobulin) are transported across pig intestinal epithelium using the InTESTine model and afterward basophil activation is measured to assess the (remaining) functional properties. Additionally, allergens are digested by pepsin prior to epithelial transport and their allergenicity is assessed in a human mast cell activation assay. Remarkably, transported Ara h 1 and 3 are not able to activate basophils, in contrast to Ara h 2 and 6. Digestion prior to transport results in a significant increase in mast cell activation of Ara h 1 and 3 dependent on the length of digestion time. Activation of mast cells by Ara h 2 and 6 is unaffected by digestion prior to transport. CONCLUSIONS: Digestion and transport influences the allergenicity of Ara h 1 and 3, but not of Ara h 2 and 6. The influence of digestion and transport on protein allergenicity may explain why current in vitro assays are not predictive for allergenicity.

Specific molten globule conformation of stem bromelain at alkaline pH.

Stem bromelain (SBM), a therapeutic protein, is rapidly absorbed across the gut epithelium. Because SBM encounters an alkaline pH at its principal site of absorption, we investigated the alkaline-induced denaturation of SBM. From pH 7 to 10, the protein's secondary structure remained the same, although a slight loss of tertiary structure was observed. Above pH 10, there was a significant and irreversible loss of secondary and tertiary structure. At pH 10, SBM showed enhanced tryptophan fluorescence, however, the number of accessible tryptophans remained the same. The thermodynamics of temperature transition at pH 7 and 10 were strikingly different, with the former showing a two-phase transition endotherm, and the latter a broad non-two-state transition. At pH 10, SBM showed a significant increase in 8-anilino-1-naphthalene-sulfonate binding relative to the native state, suggestive of a specific molten globule (SMG) state. These studies suggest a distinct conformational rearrangement in SBM, at the protein's isoelectric point.

LC-MS/MS analysis of partial structure of Panax ginseng protein and its distribution in vivo.

Protein from Panax ginseng can improve learning, memory, and analgesia. Here, we investigated a fluorescence labeling method that can be used to determine the in vivo distribution of P. ginseng protein (PGP). High-performance liquid chromatography (HPLC) was used to define the amino acid composition and molecular weight of PGP; LC-MS/MS was used to identify the PGP structure, which was fluorescently-labeled using a fluorescein isothiocyanate (FITC) probe. The connection form of the PGP fluorescent marker (PGP-FITC) was identified by ultraviolet and infrared spectrophotometry. The in vivo distribution of PGP was observed by fluorescence imaging, and tissue content was determined. Results showed that PGP was enriched in the brain and that vascular epithelial cells showed specific uptake. We provide an experimental method to label and identify the in vivo distribution of PGP, which forms the basis for future studies to determine whether PGP can penetrate the blood-brain barrier (BBB) and elucidate the transport mechanism.

The Effect of the Food Matrix on the In Vitro Bio-Accessibility and IgE Reactivity of Peanut Allergens.

SCOPE: Factors such as food processing, the food matrix, and antacid medication may affect the bio-accessibility of proteins in the gastrointestinal tract and hence their allergenic activity. However, at present they are poorly understood. METHODS AND RESULTS: Roasted peanut flour was incorporated into either a chocolate dessert or cookie matrix and bio-accessibility were assessed using an in vitro digestion system comprising a model chew and simulated gastric and duodenal digestion. Protein digestion was monitored by SDS-PAGE and immunoreactivity analyzed by immunoblotting and immunoassay. IgE reactivity was assessed by immunoassay using serum panels from peanut-allergic subjects. Roasted peanut flour proteins proved highly digestible following gastro-duodenal digestion even when incurred into a food matrix, with only low molecular weight polypeptides of Mr < 8 kDa remaining. When gastric digestion was performed at pH 6.5 (simulating the effect of antacid medication), peanut proteins are not digested; subsequent duodenal digestion is also limited. IgE reactivity of the major peanut allergens Ara h 1, Ara h 2, and Ara h 6, although reduced, was retained after oral-gastro-duodenal digestion irrespective of digestion conditions employed. CONCLUSION: Peanut allergen bio-accessibility is unaffected by the dessert or cookie matrices whilst high intra-gastric pH conditions render allergens more resistant to digestion.

Effect of pH regulation on the components and functional properties of proteins isolated from cold-pressed rapeseed meal through alkaline extraction and acid precipitation.

Cold-pressed rapeseed meal with high protein content (38.76% protein dry weight basis) was used to prepare rapeseed protein isolates (RPIs) by alkaline extraction (pH 8.0, 9.0, 10.0, 11.0, 12.0 and 13.0) and acid precipitation (pH 3.0, 3.5, 4.0, 4.5, 5.0 and 5.5). The protein with an intact structure and the highest yield (65.08%) was obtained at extraction pH 9.0 and precipitation pH 4.5, accompanied by the lowest D-amino acid content, the lightest colour and the lowest contents of glucosinolates (2.85 mmol/kg), phytic acid (1.05 mg/g) and sinapine (0.68 mg/g). Additionally, water/oil absorption, foaming and emulsifying capacities decreased with decreasing precipitation pH, while the solubility showed the reverse trend. During gastric simulation digestion, the α-polypeptide of cruciferin and napin in the RPIs showed digestive resistance. Overall, pH regulation might be an effective method to isolate high quality RPIs for use in the food processing industry.

Effect of protein aggregation in wheat-legume mixed pasta diets on their in vitro digestion kinetics in comparison to "rapid" and "slow" animal proteins.

The aim of this work was to evaluate the impact of incorporating different legume flours (faba bean, lentil or split pea flours) on the pasta protein network and its repercussion on in vitro protein digestibility, in comparison with reference dairy proteins. Kinetics and yields of protein hydrolysis in legume enriched pasta and, for the first time, the peptidomes generated by the pasta at the end of the in vitro gastric and intestinal phases of digestion are presented. Three isoproteic (21%) legume enriched pasta with balanced essential amino acids, were made from wheat semolina and 62% to 79% of legume flours (faba bean or F-pasta; lentil or L-pasta and split pea or P-pasta). Pasta were prepared following the conventional pastification steps (hydration, mixing, extrusion, drying, cooking). Amino acid composition and protein network structure of the pasta were determined along with their culinary and rheological properties and residual trypsin inhibitor activity (3-5% of the activity initially present in raw legume flour). F- and L-pasta had contrasted firmness and proportion of covalently linked proteins. F-pasta had a generally weaker protein network and matrix structure, however far from the weakly linked soluble milk proteins (SMP) and casein proteins, which in addition contained no antitrypsin inhibitors and more theoretical cleavage sites for digestive enzymes. The differences in protein network reticulation between the different pasta and between pasta and dairy proteins were in agreement in each kinetic phase with the yield of the in vitro protein hydrolysis, which reached 84% for SMP, and 66% for casein at the end of intestinal phase, versus 50% for L- and P-pasta and 58% for F-pasta. The peptidome of legume enriched pasta is described for the first time and compared with the peptidome of dairy proteins for each phase of digestion. The gastric and intestinal phases were important stages of peptide differentiation between legumes and wheat. However, peptidome analysis revealed no difference in wheat-derived peptides in the three pasta diets regardless of the digestion phase, indicating that there was a low covalent interaction between wheat gluten and legume proteins.

Birch pollen allergen Bet v 1 binds to and is transported through conjunctival epithelium in allergic patients.

BACKGROUND: Previous work in type-I pollen allergies has mainly focused on lymphocytes and immune responses. Here, we begin to analyse with a systems biology view the differences in conjunctival epithelium obtained from healthy and allergic subjects. METHODS: Transcriptomics analysis combined with light and electron microscopic analysis of birch pollen allergen Bet v 1 located within conjunctival epithelial cells and tissues from birch allergic subjects and healthy controls was carried out. RESULTS: Bet v 1 pollen allergen bound to conjunctival epithelial cells within minutes after the exposure even during the nonsymptomatic winter season only in allergic, but not in healthy individuals. Light- and electron microscopy showed that Bet v 1 was transported through the epithelium within lipid rafts/caveolae and reached mast cells only in allergic patients, but not in healthy individuals. Transcriptomics yielded 22 putative receptors expressed at higher levels in allergic epithelium compared with healthy specimens. A literature search indicated that out of these receptors, eight (i.e. 37%) were associated with lipid rafts/caveolae, which suggested again that Bet v 1 transport is lipid raft/caveola-dependent. CONCLUSIONS: We show a clear difference in the binding and uptake of Bet v 1 allergen by conjunctival epithelial cells in allergic vs healthy subjects and several putative lipid raft/caveolar receptors were identified, which could mediate or be co-transported with this entry. The application of discovery driven methodologies on human conjunctival epithelial cells and tissues can provide new hypotheses worth a further analysis to the molecular mechanisms of a complex multifactorial disease such as type-I birch pollen allergy.

Dry fractionation of surface abrasion for polyphenol-enriched buckwheat protein combined with hydrothermal treatment.

Buckwheat constitutes a good source of bioactive components. A dry fractionation of surface abrasion for polyphenol-enriched protein combined with hydrothermal treatment was evaluated as an alternative to conventional wet extraction from tartary buckwheat (Fagopyrum esculentum Moench). The protein contents and the total polyphenol contents of both free and bound polyphenol gradually decreased in the order from the outer to the inner fractions. Polyphenol-enriched buckwheat protein flour was successfully enrichment with a maximum polyphenol content of 55 mg/g. Moreover, starch digestibility and polyphenols bioaccessibility of the buckwheat protein were increased with hydrothermal treatment time, while protein digestibility decreased slightly. Besides, most of the aroma compounds increased during the hydrothermal treatment. The assessment results demonstrate that the sustainability dry surface abrasion process in combination with hydrothermal treatment should be encouraged in processing functional protein fractions and improving both qualities of end use products and health benefits.