Albumins Research Articles

Comprehensive study of chiral herbicide flusulfinam uptake, translocation, degradation, and subcellular distribution in rice (Oryza sativa L.)

The biological behavior of flusulfinam, a potential commercial chiral herbicide for rice, has not been well explored. Herein, the uptake of chiral flusulfinam by rice and its transport, degradation, and subcellular distribution in rice (Oryza sativa L.) were investigated. The enantiomeric fraction (EF) in roots was 0.54 during 0 d to 7 d in hydroponic laboratory conditions. The bioconcentration factor of flusulfinam enantiomers was 2.1, suggesting an absence of observed enantioselectivity in the absorption process. Notably, the EF in the shoots decreased to 0.35 on the 7th day. The translocation factors of R- and S-flusulfinam were 0.12 and 0.27, respectively, indicating a preferential transfer of the S-flusulfinam from the root to the shoot. Flusulfinam was identified in the root after spraying. The translocation factors of R- and S-flusulfinam were consistently similar, signifying the capacity for downward movement without enantioselectivity. Interestingly, the degradation half-lives of R- and S-flusulfinam in the total plant were 5.50 and 5.06 d (p < 0.05), respectively, supporting the preferential degradation of S-flusulfinam throughout the total plant. Flusulfinam primarily entered the roots via the apoplastic pathway and was subsequently transported within the plant through aquaporins and ion channels. The subcellular distribution experiment revealed the predominant accumulation of flusulfinam enantiomers in soluble components (84%) with no enantioselectivity in these processes. There was upregulation lipid transfer protein-2 and carboxylesterases15 genes, which could explain the preferential transport and degradation of S-flusulfinam. This study is important in assessing the environmental risk associated with flusulfinam and ensuring food safety.

The Long-Distance Transport of Jasmonates in Salt-Treated Pea Plants and Involvement of Lipid Transfer Proteins in the Process.

The adaption of plants to stressful environments depends on long-distance responses in plant organs, which themselves are remote from sites of perception of external stimuli. Jasmonic acid (JA) and its derivatives are known to be involved in plants' adaptation to salinity. However, to our knowledge, the transport of JAs from roots to shoots has not been studied in relation to the responses of shoots to root salt treatment. We detected a salt-induced increase in the content of JAs in the roots, xylem sap, and leaves of pea plants related to changes in transpiration. Similarities between the localization of JA and lipid transfer proteins (LTPs) around vascular tissues were detected with immunohistochemistry, while immunoblotting revealed the presence of LTPs in the xylem sap of pea plants and its increase with salinity. Furthermore, we compared the effects of exogenous MeJA and salt treatment on the accumulation of JAs in leaves and their impact on transpiration. Our results indicate that salt-induced changes in JA concentrations in roots and xylem sap are the source of accumulation of these hormones in leaves leading to associated changes in transpiration. Furthermore, they suggest the possible involvement of LTPs in the loading/unloading of JAs into/from the xylem and its xylem transport.

Recombinant production of Morus alba lipid transfer protein and prediction of its allergenic epitopes

BackgroundWhite mulberry (Morus alba) pollen grains trigger allergic diseases including asthma, allergic rhinitis, and allergic conjunctivitis. Non-specific lipid transfer proteins (nsLTPs) are robust allergens found in higher plants. Although it was isolated and immunologically characterized in the fruits of Morus nigra, it has not yet been determined whether nsLTP is one of the M. alba pollen allergens. ObjectiveThis study aims to examine the possible allergenicity of M. alba pollen nsLTP by producing it recombinantly, followed by immunoblotting. MethodsTotal RNA was isolated from M. alba pollen grains, and the nsLTP gene was obtained by reverse transcriptase polymerase reaction. It was cloned into Escherichia coli using pQE2 plasmid and purified by immobilized metal affinity chromatography following the expression. To confirm the immunoreactivity of the purified r-nsLTP, immunoblotting was performed with the serum of patients allergic to M. alba pollen. B-cell epitopes of nsLTP were also predicted using the bioinformatics tools, namely Bioinformatics Predicted Antigenic Peptides, BepiPred-2.0, and the Immune Epitope Database, whereas T-cell epitopes were estimated by using NetMHCIIpan-4.0 and NetMHCII-2.3 servers. ResultsThe r-nsLTP showed positive results in immunoblotting with sera of 7/7 (100%) of M. alba allergic patients. Five B-cell epitopes (26–31, 43–50, 65–71, 92–100, 105–115) and three T-cell epitopes (57–65, 78–86, 105–113) were predicted by in silico analyses. ConclusionData obtained in this study are expected to support the use of r-nsLTP in solutions for diagnosis and treatment of M. alba allergy and cross-reactivities between nsLTPs, as well as recombinant allergen production for vaccination.

Food-Pollen Cross-Reactivity and its Molecular Diagnosis in China.

Plant-derived foods are one of the most common causative sources of food allergy in China, with a significant relationship to pollinosis. This review aims to provide a comprehensive overview of this food-pollen allergy syndrome and its molecular allergen diagnosis to better understand the cross-reactive basis. Food-pollen cross-reactivity has been mainly reported in Northern China, Artemisia pollen is the major related inhalant source, followed by tree pollen (Betula), while grass pollen plays a minor role. Pollen allergy is relatively low in Southern China, with allergies to grass pollen being more important than weed and tree pollens. Rosaceae fruits and legume seeds stand out as major related allergenic foods. Non-specific lipid transfer protein (nsLTP) has been found to be the most clinically relevant cross-reacting allergenic component, able to induce severe reactions. PR-10, profilin, defensin, chitinase, and gibberellin-regulated proteins are other important cross-reactive allergen molecules. Artemisia pollen can induce allergenic cross-reactions with a wide range of plant-derived foods in China, and spring tree pollens (Betula) are also important. nsLTP found in both pollen and plant-derived food is considered the most significant allergen in food pollen cross-reactivity. Component-resolved diagnosis with potential allergenic proteins is recommended to improve diagnostic accuracy and predict the potential risk of causing allergic symptoms.

Structural and Functional Characterization of New Lipid Transfer Proteins with Chitin-Binding Properties: Insights from Protein Structure Prediction, Molecular Docking, and Antifungal Activity.

Faced with the emergence of multiresistant microorganisms that affect human health, microbial agents have become a serious global threat, affecting human health and plant crops. Antimicrobial peptides have attracted significant attention in research for the development of new microbial control agents. This work's goal was the structural characterization and analysis of antifungal activity of chitin-binding peptides from Capsicum baccatum and Capsicum frutescens seeds on the growth of Candida and Fusarium species. Proteins were initially submitted to extraction in phosphate buffer pH 5.4 and subjected to chitin column chromatography. Posteriorly, two fractions were obtained for each species, Cb-F1 and Cf-F1 and Cb-F2 and Cf-F2, respectively. The Cb-F1 (C. baccatum) and Cf-F1 (C. frutescens) fractions did not bind to the chitin column. The electrophoresis results obtained after chromatography showed two major protein bands between 3.4 and 14.2 kDa for Cb-F2. For Cf-F2, three major bands were identified between 6.5 and 14.2 kDa. One band from each species was subjected to mass spectrometry, and both bands showed similarity to nonspecific lipid transfer protein. Candida albicans and Candida tropicalis had their growth inhibited by Cb-F2. Cf-F2 inhibited the development of C. albicans but did not inhibit the growth of C. tropicalis. Both fractions were unable to inhibit the growth of Fusarium species. The toxicity of the fractions was tested in vivo on Galleria mellonella larvae, and both showed a low toxicity rate at high concentrations. As a result, the fractions have enormous promise for the creation of novel antifungal compounds.

Tolerance of peanuts and tree nuts in Spanish children with exclusive sensitization to lipid transfer proteins.

Allergy to peanuts and tree nuts is a common cause of food allergy in Spain, with lipid transfer proteins (LTP) being the most frequently recognized panallergen. LTP sensitization often leads to multiple food group sensitivities, resulting in overly restrictive diets that hinder patient's quality of life. This study aimed to assess the tolerance of peanuts and tree nuts (hazelnuts and walnuts) in children sensitized to LTP, potentially mitigating the need for such diets. This prospective study enrolled individuals diagnosed with allergy to peanuts, hazelnuts, or walnuts. Data were collected from medical records, including demographics and clinical history. Allergological assessment comprised skin prick tests using commercial extracts and the nuts in question, alongside measurements of total and specific IgE to nuts and their primary molecular components. Participants showing positive LTP sensitization without sensitization to seed storage proteins underwent open oral nut challenges. A total of 75 individuals labeled as allergic to peanuts, 44 to hazelnuts, and 51 to walnuts were included. All of them underwent an open oral provocation test with the incriminated nut, showing a high tolerance rate. Peanut was tolerated by 98.6% of patients, 97.72% tolerated hazelnut, and 84.3% tolerated walnut. The findings suggest that the majority of patients allergic to peanuts, hazelnuts, or walnuts, due to LTP sensitization and lacking IgE reactivity to seed storage proteins, can tolerate these nuts. This supports the need for personalized nut tolerance assessments to avoid unnecessary dietary restrictions.

Clinical and serological characteristics of patients allergic to LTP.

Allergy to lipid transfer proteins (LPT) is common in Mediterranean Europe, and it causes severe reactions in patients and affects multiple foods, impairing the quality of life. This study aimed to describe the clinical and sensitization profile of patients with LTP syndrome and to determine a clinical pattern of severity. Molecular diagnosis is shown in a broad population through microarrays. This study was performed at the LTP Allergy Consultation of the Reina Sofia Hospital in Murcia, Spain. We analyzed the patients' characteristics, reactions, cofactors, food implicated, quality of life, skin prick test to food and aeroallergens, and serologic parameters, such as total immunoglobulin E, peach LTP (Pru p 3 IgE) and immunoglobulin G4, and microarray Immuno Solid-phase Allergen Chip (ISAC). We related the severity of the reactions with other variables. We presented a series of 236 patients diagnosed with LTP allergy, 54.66% suffering from anaphylaxis, 36.02% from urticaria angioedema, and 9.32% from oral allergy syndrome. The most frequently implicated food was peach, producing symptoms in 70% of patients, followed by walnut in 55%, peanut in 45%, hazelnut in 44%, and apple in 38% patients. Regarding the food that provoked anaphylaxis, walnut was the most frequent instigator, along with peach, peanut, hazelnut, almond, sunflower seed, and apple. According to the severity of LPT reaction, we did not discover significant differences in gender, age, food group involved, and serologic parameters. We found differences in the presence of cofactors, with 48.84% of cofactors in patients with anaphylaxis, compared to 27.1% in patients without anaphylaxis and in family allergy background (P < 0.0001). In our series of patients, 54% presented anaphylaxis, and the foods that most frequently produced symptoms were peaches, apples, and nuts. Cofactors and family allergy backgrounds were associated with the severity of LPT reaction.

IgE-mediated reactivity to non-specific lipid transfer protein (nsLTP): clinical implications and management ‒ a 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.

Nonspecific lipid-transfer proteins trigger TLR2 and NOD2 signaling and undergo ligand-dependent endocytosis in epithelial cells

BackgroundAllergens can cross the epithelial barrier to enter the body but how this cellular passage affects protein structures and the downstream interactions with the immune system are still open questions. ObjectiveWe show the molecular details and the effects of three non-specific lipid transfer proteins (nsLTPs; Mal d 3, Cor a 8 and Pru p 3) upon epithelial cell uptake and transport. MethodsWe used fluorescent imaging, flow cytometry, proteomic and lipidomic screenings to identify the mechanism involved in nsLTP cellular uptake and signaling on selected epithelial and transgenic cell lines. ResultsNsLTPs are transported across the epithelium without affecting cell membrane stability or viability and allergen uptake was largely impaired by inhibition of clathrin-mediated endocytosis (CME). Analysis of the lipidome associated with nsLTPs showed a wide variety of lipid ligands predicted to bind inside the allergen hydrophobic cavity. Importantly, the internalization of nsLTPs was contingent upon these ligands in the protein complex.nsLTPs were found to initiate cellular signaling via TLR2 but not the CD1d receptor, despite neither being essential for nsLTP endocytosis. We also provide evidence that the three allergens induced intracellular stress signaling through activation of the NOD2 pathway. ConclusionsOur work consolidates the current model on nsLTP-epithelial cell interplay and adds molecular details about cell transport and signaling. Additionally, we have developed a versatile toolbox to extend these investigations to other allergens and cell types.

Metabolic channeling of lipids via the contact zones between different organelles.

Various lipid transfer proteins (LTPs) mediate the inter-organelle transport of lipids. By working at membrane contact zones between donor and acceptor organelles, LTPs achieve rapid and accurate inter-organelle transfer of lipids. This article will describe the emerging paradigm that the action of LTPs at organelle contact zones generates metabolic channeling events in lipid metabolism, mainly referring to how ceramide synthesized in the endoplasmic reticulum is preferentially metabolized to sphingomyelin in the distal Golgi region, how cholesterol and phospholipids receive specific metabolic reactions in mitochondria, and how the hijacking of host LTPs by intracellular pathogens may generate new channeling-like events. In addition, the article will discuss how the function of LTPs is regulated, exemplified by a few representative LTP systems, and will briefly touch on experiments that will be necessary to establish the paradigm that LTP-mediated inter-organelle transport of lipids is one of the mechanisms of compartmentalization-based metabolic channeling events.

Proteomic Characterization of a Lunasin-Enriched Soybean Extract Potentially Useful in the Treatment of Helicobacter pylori Infection.

Helicobacter pylori infection affects over 50% of the world's population and leads to chronic inflammation and gastric disorders, being the main pathogen correlated to gastric cancer development. Increasing antibiotic resistance levels are a major global concern and alternative treatments are needed. Soybean peptides and other compounds might be an alternative in the treatment to avoid, eradicate and/or control symptoms of H. pylori infection. This study aimed to characterize a lunasin-enriched soybean extract (LSE) using proteomics tools and to evaluate its antioxidant, anti-inflammatory and antibacterial properties against H. pylori infection. By LC-MS/MS analysis, 124 proteins were identified, with 2S albumin (lunasin and large-chain subunits) being the fourth most abundant protein (8.9%). Lunasin consists of 44 amino acid residues and an intramolecular disulfide bond. LSE at a low dose (0.0625 mg/mL) reduced ROS production in both H. pylori-infected and non-infected AGS gastric cells. This led to a significant reduction of 6.71% in the levels of pro-inflammatory interleukin (IL)-8. LSE also showed antibacterial activity against H. pylori, which can be attributed to other soybean proteins and phenolic compounds. Our findings suggest that LSE might be a promising alternative in the management of H. pylori infection and its associated symptoms.

Isolation, Characterization and IgE Binding of Two 2S Albumins of Pomegranate Seeds.

Literature reports suggest that the presence of proteins in pomegranate seeds is responsible for sensitization and IgE-mediated allergic reactions. The objective of this study was the analysis of a pomegranate seed extract and the isolation and characterization of proteins contained in high amounts. The extract characterization showed a protein profile with main bands at about 18 kDa and below 10 kDa upon SDS-PAGE, and molecules were recognized by specific IgEs upon immunoblotting. Then, two new 2S albumins, a monomeric and a heterodimeric one, were isolated by using classical biochemical methods. They were identified via direct protein sequencing and mass spectrometry, and their primary structure was analyzed and compared with homologous allergenic proteins via bioinformatics. In an Italian population of 703 suspected allergic patients, analyzed by using the FABER® test, the frequency of sensitization to the monomeric and heterodimeric 2S albumins was 1.7% and 0.28%, respectively. This study reports for the first time the isolation and characterization of two 2S albumins from pomegranate seeds. The clinical relevance of these molecules needs further investigation, for instance in populations having different exposures and allergy profiles.

Differential expression and bioinformatics analysis of proteins in response to NaCl stress in purslane

Proteomics serves as a crucial method for elucidating plant stress resistance mechanisms under environmental pressures. Purslane (Portulaca oleracea), valued for its medicinal, edible, and halophytic properties, holds significant nutritional importance with diverse applications. Despite its importance, limited studies have explored the salt tolerance mechanisms in purslane. Differential protein expression in two purslane genotypes (‘PL’ and ‘LCL’) under NaCl stress were analyzed using tandem mass tag technology. The function of differentially expressed proteins was analyzed using bioinformatics tools. According to KEGG and COG analysis, carbohydrate metabolism and lipid metabolism were the most important metabolite pathways in purslane under salt stress. Compared with control, 48 up-regulated and 43 down-regulated proteins in ‘PL’ and 22 up-regulated and 26 down-regulated proteins in ‘LCL’ were found under 72 h of 200 mM NaCl stress. Six proteins were up-regulated and six down-regulated in both genotypes due to salt stress. Among them, four up-regulated proteins and two down-regulated proteins were related to carbohydrate metabolism. Specific protein expressions in carbohydrate metabolism pathway may be the vital reason of salt-tolerance in purslane. The differential protein expression in two purslane genotypes under salt stress unveiled significant up-regulation of seven proteins belonging to the ribosomal protein family, along with two non-specific lipid-transfer proteins and two lipid transfer-like proteins in the ‘PL’ genotype. Additionally, the down-regulation of relatively few photosynthesis-related proteins were observed in the ‘PL’ genotype. Differential expression of these proteins might be associated with the salt tolerance in the salt-tolerant variety.

Aryl amino acetamides prevent Plasmodium falciparum ring development via targeting the lipid-transfer protein PfSTART1

With resistance to most antimalarials increasing, it is imperative that new drugs are developed. We previously identified an aryl acetamide compound, MMV006833 (M-833), that inhibited the ring-stage development of newly invaded merozoites. Here, we select parasites resistant to M-833 and identify mutations in the START lipid transfer protein (PF3D7_0104200, PfSTART1). Introducing PfSTART1 mutations into wildtype parasites reproduces resistance to M-833 as well as to more potent analogues. PfSTART1 binding to the analogues is validated using organic solvent-based Proteome Integral Solubility Alteration (Solvent PISA) assays. Imaging of invading merozoites shows the inhibitors prevent the development of ring-stage parasites potentially by inhibiting the expansion of the encasing parasitophorous vacuole membrane. The PfSTART1-targeting compounds also block transmission to mosquitoes and with multiple stages of the parasite’s lifecycle being affected, PfSTART1 represents a drug target with a new mechanism of action.

Molecular Biomarkers of Canine Reproductive Functions.

The aim of the current study is to review potential molecular biomarker substances selected so far as useful for assessing the quality of dog semen. Proteins, lipids, carbohydrates, and ions can serve as molecular biomarkers of reproductive functions (BRFs) for evaluating male reproductive health and identifying potential risk factors for infertility or reproductive disorders. Evaluation of BRF levels in semen samples or reproductive tissues may provide insights into the underlying causes of infertility, such as impaired sperm function, abnormal sperm-egg interaction, or dysfunction of the male reproductive tract. Molecular biomarker proteins may be divided into two groups: proteins that are well-studied, such as A-kinase anchoring proteins (AKAPs), albumins (ALBs), alkaline phosphatase (ALPL), clusterin (CLU), canine prostate-specific esterase (CPSE), cysteine-rich secretory protein 2 (CRISP2), lactotransferrin (LTF), metalloproteinases (MMPs), and osteopontin (OPN) and proteins that are not well-studied. Non-protein markers include lipid-based substances (fatty acids, phosphatidylcholine), carbohydrates (glycosaminoglycans), and ions (zinc, calcium). Assessing the levels of BRFs in semen samples may provide valuable information for breeding management and reproductive assessments in dogs. This review systematizes current knowledge that could serve as a starting point for developing practical tests with the use of biomarkers of canine reproductive functions and their predictive value for assisted reproductive technique outcomes and semen preservation.

Non-Specific Lipid Transfer Protein Amb a 6 Is a Source-Specific Important Allergenic Molecule in Ragweed Pollen.

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.

The role of the 7S/11S globulin ratio in the gelling properties of mixed β-lactoglobulin/pea proteins systems

Using a combination of pea protein isolates (PPI) and β-lactoglobulin (βlg) presents an interesting approach to improve the gelling properties of PPI compared to the use of PPI alone. While controlling the 7S/11S ratio has proven effective in improving the gelling properties of PPI, more complex models need to be studied. Specifically, the effects of diverse 7S/11S ratios and the inclusion of βlg and 2S albumin on gel formation remain unexplored. Therefore, this study aimed to 1) determine the optimal 7S/11S ratios and 2) identify the interactions involved in the formation of model βlg—pea protein gels generated from globulin and albumin-enriched fractions. Results revealed that pea protein gels with a 7S/11S ratio of ∼ 1.89 achieved the highest firmness (131 Pa). Gelation was primarily driven by hydrophobic interactions (∼ 41%) involving 7S vicilin and 2S albumin, while most of the 11S legumin did not contribute to the protein gel. Conversely, a 7S/11S ratio of ≤ 1 facilitated the formation of the firmest gels (300–657 Pa) in the presence of βlg. These gels were primarily formed via disulfide bonds (36–65%) with βlg, 11S legumin, and 2S albumin as the main proteins involved, while 7S vicilin gelled independently through hydrophobic interactions. Based on these results, we propose a mechanism illustrating the interactions in mixed βlg—pea protein gels. This study provides new insights into how 7S/11S ratios, gel firmness, and protein interactions interplay during the gelation of mixed βlg–pea protein systems, paving the way for innovation in developing a new food category.

Deciphering the drug delivery potential of Type1 lipid transfer protein from Citrus sinensis for enhancing the therapeutic efficacy of drugs

Type1 Non-specific Lipid Transfer Protein (CsLTP1) from Citrus sinensis is a small cationic protein possessing a long tunnel-like hydrophobic cavity. CsLTP1 performing membrane trafficking of lipids is a promising candidate for developing a potent drug delivery system. The present work includes in-silico studies and the evaluation of drugs binding to CsLTP1 using biophysical techniques along with the investigation of CsLTP1's ability to enhance the efficacy of drugs employing cell-based bioassays. The in-silico investigations identified Panobinostat, Vorinostat, Cetylpyridinium Chloride, and Fulvestrant with higher affinities and stability of binding to the hydrophobic pocket of CsLTP1. SPR studies revealed strong binding affinities of anticancer drugs, Panobinostat (KD = 1.40 μM) and Vorinostat (KD = 2.17 μM) to CsLTP1 along with the binding and release kinetics. CD and fluorescent spectroscopy revealed drug-induced conformational changes in CsLTP1. CsLTP1-associated drug forms showed remarkably enhanced efficacy in MCF-7 cells, representing increased cell cytotoxicity, intracellular ROS, reduced mitochondrial membrane potential, and up-regulation of proapoptotic markers than the free drugs employing qRT-PCR and western blot analysis. The findings demonstrate that CsLTP1 binds strongly to hydrophobic drugs to facilitate their transport, hence improving their therapeutic efficacy revealed by the in-vitro investigations. This study establishes an excellent foundation for developing CsLTP1-based efficient drug delivery system.

Bioactive peptides released by lactic acid bacteria fermented pistachio beverages

Fruit and vegetable are the raw materials for many popular fermented beverages with potential health benefits. In the present study, a novel pistachio beverage fermented with selected strains of lactic acid bacteria belonging to the species Leuconostoc pseudomesenteroides and Companilactobacillus paralimentarius was produced, and the extent of proteolysis was evaluated. Physico-chemical and microbiological analyses, together with peptidomics and proteomics, were carried out on the beverage after 24 h of fermentation, using both the non-inoculated and chemically acidified beverage as controls. Amino acid sequence of hundreds of peptides mainly released from 2S albumin, 11S and 7S globulin were characterized. The number and frequency of identified peptides were higher in beverage started with Leuconostoc pseudomesenteroides, followed by Companilactobacillus paralimentarius and acidic beverage. These results suggest that, in addition to endogenous proteases active at acidic pH, the proteolytic system of LAB directly participated to peptide degradation to some extent. According to the BIOPEP database, a group of 31 peptides were potentially bioactive, and primarily associated with antioxidative properties, ACE and DPP-IV inhibition. The beverage started with Leuconostoc pseudomesenteroides showed the highest amount and number of bioactive peptides. This study lays the foundations for the design of novel pistachio-fermented beverage with potential health properties.

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.