Modification of the physicochemical, functional, biochemical and structural properties of a soursop seed (Annona muricata L.) protein isolate treated with high-intensity ultrasound.

The obtained seeds from fruit processing are considered by-products containing proteins that could be utilized as ingredients in food manufacturing. However, in the specific case of soursop seeds, their usage for the preparation of protein isolates is limited. In this investigation a protein isolate from soursop seeds (SSPI) was obtained by alkaline extraction and isoelectric precipitation methods. The SSPI was sonicated at 200, 400 and 600 W during 15 and 30 min and its effect on the physicochemical, functional, biochemical, and structural properties was evaluated. Ultrasound increased (p < 0.05) up to 5 % protein content, 261 % protein solubility, 60.7 % foaming capacity, 30.2 % foaming stability, 86 % emulsifying activity index, 4.1 % emulsifying stability index, 85.4 % in vitro protein digestibility, 423.4 % albumin content, 83 % total sulfhydryl content, 316 % free sulfhydryl content, 236 % α-helix, 46 % ß-sheet, and 43 % ß-turn of SSPI, in comparison with the control treatment without ultrasound. Furthermore, ultrasound decreased (p < 0.05) up to 50 % particle size, 37 % molecular flexibility, 68 % surface hydrophobicity, 41 % intrinsic florescence spectrum, and 60 % random coil content. Scanning electron microscopy analysis revealed smooth structures of the SSPI with molecular weights ranging from 12 kDa to 65 kDa. The increase of albumins content in the SSPI by ultrasound was highly correlated (r = 0.962; p < 0.01) with the protein solubility. Improving the physicochemical, functional, biochemical and structural properties of SSPI by ultrasound could contribute to its utilization as ingredient in food industry.

Subcellular localization of PD-L1 and cell-cycle-dependent expression of nuclear PD-L1 variants: implications for head and neck cancer cell functions and therapeutic efficacy.

The programmed cell death 1 ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) axis is primarily associated with immunosuppression in cytotoxic T lymphocytes (CTLs). However, mounting evidence is supporting the thesis that PD-L1 not only functions as a ligand but mediates additional cellular functions in tumor cells. Moreover, it has been demonstrated that PD-L1 is not exclusively localized at the cellular membrane. Subcellular fractionation revealed the presence of PD-L1 in various cellular compartments of six well-characterized head and neck cancer (HNC) cell lines, including the nucleus. Via Western blotting, we detected PD-L1 in its well-known glycosylated/deglycosylated state at 40-55 kDa. In addition, we detected previously unknown PD-L1 variants with a molecular weight at approximately 70 and > 150 kDa exclusively in nuclear protein fractions. These in vitro findings were confirmed with primary tumor samples from head and neck squamous cell carcinoma (HNSCC) patients. Furthermore, we demonstrated that nuclear PD-L1 variant expression is cell-cycle-dependent. Immunofluorescence staining of PD-L1 in different cell cycle phases of synchronized HNC cells supported these observations. Mechanisms of nuclear PD-L1 trafficking remain less understood; however, proximity ligation assays showed a cell-cycle-dependent interaction of the cytoskeletal protein vimentin with PD-L1, whereas vimentin could serve as a potential shuttle for nuclear PD-L1 transportation. Mass spectrometry after PD-L1 co-immunoprecipitation, followed by gene ontology analysis, indicated interaction of nuclear PD-L1 with proteins involved in DNA remodeling and messenger RNA (mRNA) splicing. Our results in HNC cells suggest a highly complex regulation of PD-L1 and multiple tumor cell-intrinsic functions, independent of immune regulation. These observations bear significant implications for the therapeutic efficacy of immune checkpoint inhibition.

Microwave radiation induces modifications in the protein fractions of tef flours and modulates their derived techno-functional properties.

The impact of microwave (MW) treatments on the structure, solubility, and techno-functional properties of the proteins in starchy matrices is still poorly understood. This study aimed to investigate the effects of MW intensity by applying 1, 2, and 6 min of radiation on two tef flour varieties moistened at 15 % and 25 %. The fractionation method recovered ∼83 % of the total protein content in untreated flours. The interaction between treatment time and moisture content (MC) significantly influenced the extraction of protein fractions. Samples treated at 25 %MC showed significant reductions in albumins (up to -74 %), globulins (up to -79 %), and prolamins (up to -32 %). The SDS-extractable proteins of both tef flours presented similar molecular weights (12-100 kDa). SDS-PAGE analysis revealed decreased band intensity in MW-treated samples compared to untreated flours, and confocal analysis showed changes in the native state of proteins in treated samples. Shorter treatments at low MC significantly improved the emulsifying stability of tef flours, particularly in brown tef flour, with an enhancement of up to 203 %. The hydration properties significantly increased in flours treated at 25 %MC for 6 min. Pearson correlation analysis demonstrated the influence of treatment time and MC on protein recovery and functional properties of tef flours.

Distinct myofibrillar sub-proteomic profiles are associated with the instrumental texture of aged pork loin.

Fresh pork tenderness contributes to consumer satisfaction with the eating experience. Postmortem proteolysis of proteins within and between myofibrils has been closely linked with pork tenderness development. A clear understanding of the molecular features associated with pork tenderness development will provide additional targets and open the door to new solutions to improve and make pork tenderness development more consistent. Therefore, the objective was to utilize liquid chromatography and mass spectrometry with tandem mass tag (TMT) multiplexing to evaluate myofibrillar sub-proteome differences between pork chops of different instrumental star probe values. Pork loins (N = 120) were collected from a commercial harvest facility at 24 h postmortem. Quality and sensory attributes were evaluated at 24 h postmortem and after ~2 weeks of postmortem aging. Pork chops were grouped into 4 groups based on instrumental star probe value (group A,x¯â€…= 4.23 kg, 3.43 to 4.55 kg; group B,x¯â€…= 4.79 kg, 4.66 to 5.00 kg; group C,x¯â€…= 5.43 kg, 5.20 to 5.64 kg; group D,x¯â€…= 6.21 kg, 5.70 to 7.41 kg; n = 25 per group). Myofibrillar proteins from the samples aged ~2 wk were fractionated, washed, and solubilized in 8.3 M urea, 2 M thiourea, and 1% dithiothreitol. Proteins were digested with trypsin, labeled with 11-plex isobaric TMT reagents, and identified and quantified using a Q-Exactive Mass Spectrometer. Between groups A and D, 54 protein groups were differentially abundant (adjusted P < 0.05). Group A had a greater abundance of proteins related to the thick and thin filament and a lesser abundance of Z-line-associated proteins and metabolic enzymes than group D chops. These data highlight that distinct myofibrillar sub-proteomes are associated with pork chops of different tenderness values. Future research should evaluate changes immediately and earlier postmortem to further elucidate myofibrillar sub-proteome differences over the postmortem aging period.

A primary goal of meat production is to efficiently produce safe, high-quality products. Competing interests within the goal complicate this seemingly simple aspiration. Consequently, it is necessary to emphasize efforts to enhance our comprehension of biological and molecular factors that influence quality, safety, and efficient meat production. This experiment aimed to define the proteomic profiles of the myofibrillar fraction of fresh pork with differing quality traits. Myofibrils from aged pork chops with a range of tenderness levels were used to achieve this objective. Fifty-four proteins were differentially abundant between the divergent tenderness groups. This was due to the expression profile of proteins in muscle and/or changes in proteins in the myofibrillar fraction during postmortem aging. These results inform and direct the development of antemortem and postmortem applications to ensure success in producing high-quality pork.

Bamboo forage in Peruvian Amazon: a potential feed for cattle.

During the dry and rainy seasons of the Northeastern Zone of Peru, a chemical characterization of five species of bamboo prevalent in the area (Guadua lynnclarkiae, G. takahashiae, Bambusa vulgaris, G. weberbaueri, and Dendrocalamus asper) was conducted. Then, the effect of supplementing bamboo leaves (0, 20, and 40% inclusion of D. asper) on the intake and live weight gain of 18 Gyr × Holstein heifers was evaluated for 28 days. Among the species evaluated, D. asper has the greatest crude protein (CP) concentration (158-166 g/kg Dry matter- DM), post-ruminal CP supply (127 g/kg DM), and in vitro organic matter digestibility (444-456 g/kg DM) but similar concentrations of crude ash (124 g/kg DM), calcium (2.4-2.8 mg/g), phosphorus (0.7-2.1 mg/g), protein fractions A, B1, B2, B3, C (45, 5, 35, 56, and 17g/kg DM, respectively), rumen-undegraded CP (31% CP), neutral detergent fiber (NDF, 685g/kg DM), and acid detergent fiber (ADF, 357 g/kg DM) than the other species evaluated. Dry matter intake was higher in the control treatment and in the 20% bamboo leaf inclusion treatment than in the 40% bamboo inclusion treatment. Intake of CP and NDF decreased with the increase in bamboo inclusion. Despite the differences in DM, CP, and NDF intake, the live weight gain remained similar across treatments. However, there was a greater feed conversion in the 20% bamboo leaf inclusion treatment. During the dry season, bamboo leaves can be used as an alternative supplement at a maximum inclusion of 20% without affecting the live weight gain.

Characterization of Native COMPASS Complex in Urothelial Carcinoma Cells by Size Exclusion Chromatography.

The human COMPASS complexes regulate gene expression during development and cell differentiation. Three distinct subunits, KMT2C, KMT2D, and KDM6A (also known as UTX), are frequently mutated in urothelial carcinoma, possibly disrupting the formation of functional COMPASS complexes. Here, we describe methods to evaluate the formation of these large native protein complexes in urothelial carcinoma (UC) cell lines harboring different mutations in KMT2C/D. To this end COMPASS complexes were purified from nuclear extracts by size exclusion chromatography (SEC) using a Sepharose 6 column. SEC fractions were then separated by 3-8% Tris-acetate gradient polyacrylamide gel and the COMPASS complex subunits KMT2C, UTX, WDR5, and RBBP5 were detected by immunoblotting. In this fashion, the formation of a COMPASS complex could be observed in UC cells with wild-type but not in cells with mutant KMT2C and KMTD.

Nutritional and nutraceutical potential of rice bean (Vigna umbellata) -a legume with hidden potential.

In the recent years there has been paradigm shift in global agriculture for the exploration of different underutilized crops as future potential crops. Rice bean [Vigna umbellata (Thunb.) Ohwi and Ohashi] one of the lesser known pulses among Vigna species has gained attention during last decade as food and nutritional security crop. Rice bean seeds are well-balanced source of beneficial constituents such as protein, carbohydrates, minerals, vitamins, polyunsaturated fatty acids (PUFAs) and anti-oxidants for health benefits and combating malnourishment in human. In the present investigation, seeds of 15 diverse rice bean accessions from north-western Himalayan region were analyzed for nutrients, anti-nutrients and nutraceutical traits. Significant differences were observed among genotypes for different traits. The rice bean genotypes revealed variation for major quality traits including total carbohydrates (50.56-56.87%), crude protein content (22.56-25.97%) and lipid content (1.87 to 3.17%) with the higher proportion of linolenic acid followed by linoleic acid which are nutritionally desirable PUFAs. The genotype IC-548758 revealed higher proportion of desirable quality traits. Among protein fractions, globulins and albumins constituted major seed storage protein fraction in rice bean seeds. The wide range variation was also observed for anti-nutrients like including raffinose family oligosaccharides (RFOs), phenolics, tannins, trypsin inhibitor (TI), phytic acid, lipoxygenase activity and saponin content among genotypes. Insignificant correlation among iron, zinc, magnesium and manganese revealed good selection accuracy for genetic biofortification program in rice bean. In summary, the genotype IC-548757, IC-548760 and IC-548770 revealed lower proportion of anti-nutrients, whereas, the genotype IC-548759 and IC-548757 revealed higher level of free radical scavenging activity indicating nutritional and nutraceutical superiority of these genotypes. Overall, the study revealed nutritional superiority of genotype IC-548770, IC-548758 and IC-548760 with balanced proportions of nutrients and anti-nutrients. Rice bean legume has the potential to support more sustainable and resilient food and nutritional security in future. Our study highlights the potential of different rice bean genotypes as functional ingredients for future food and nutritional security programmes.

Ultrafiltration of Black Soldier Fly (Hermetia illucens) and Mealworm (Tenebrio molitor) Protein Concentrates to Enhance Emulsifying and Foaming Properties.

Mealworm, TM (Tenebrio molitor), and black soldier fly, BSF (Hermetia illucens) are of special interest for food and feed applications due to their environmental benefits such as low water and land requirements, low greenhouse gas emissions, and high feed-conversion efficiency. This study assesses the use of ultrafiltration (UF) to fractionate protein concentrates from TM and BSF (TMPC, BSFPC) in order to enhance emulsifying and foaming properties. A 30 kDa regenerated cellulose acetate membrane enabled the separation of concentrate and permeate fractions for both insect proteins from two different initial feed concentrations (10 and 7.5 g/L). Permeate flux and protein transmission behave differently depending on the insect type and the initial concentration; while for TMPC permeate flux increases with a decrease in the initial protein concentration, it is not affected for BSFPC. The existing membrane cleaning protocols are suitable for recovering water flux after UF of insect proteins, enabling membrane re-use. Emulsifying activity is maintained for all the TMPC fractions, but it is significantly lower for the permeate fractions of BSFPC. Foaming properties are maintained for all the UF fractions of BSFPC and the ones from 7.5 g/L TMPC. Acidic solubilization leads to a fraction with enhanced emulsifying capacity and one with higher foaming capacity than the original for BSFPC. This study opens the door to membrane technology for insect protein fractionation, which has not been studied so far and has already provided useful solutions for other animal and plant proteins.

Acute phase proteins levels in horses, after a single carbohydrate overload, associated with cecal alkalinization.

Introduction: Horses submitted to carbohydrate overload can develop laminitis due to changes in cecal pH and microbiota, followed by an increase in transmural absorption of luminal content, including bacterial toxins. In response to acute injury there is hepatic overproduction of several proteins known as acute phase proteins (APP). Few studies have evaluated protein fractionation to characterize the inflammatory response in acute laminitis. The aim of this study was to test the viability of an experimental model to induce acute laminitis, using a single carbohydrate overload, and the influence of a buffering solution on the development of the disease; also, study the kinetics of APP during acute laminitis, as well as the correlation between these proteins and clinical signs associated to this syndrome. Methods: Ten healthy horses were divided in a factorial and randomized way into four groups (n = 5): control group (CG), starch group (SG), buffer group (BG), and starch C buffer group (SBG). They were evaluated at seven times (T0h, T4h, T8h, T12h, T24h, T48h, and T72h), which included clinical evaluation and blood sample collection. Total serum protein and albumin concentrations were determined by colorimetry and the other APP by polyacrylamide gel electrophoresis containing sodium dodecyl sulfate and commercial ELISA kits. Data were analyzed by two-way ANOVA, followed by Tukey's test (p < 0.05). The correlation between clinical signs and APP were verified using the Pearson's correlation coefficient. Results and discussion: 40% of the animals from SG and 60% from SBG developed clinical laminitis. A single administration of buffer solution was not able to prevent clinical signs of laminitis. There was no difference between groups on total serum protein, albumin, serum amyloid A and C-reactive protein concentrations (p > 0.05). Transferrin, considered a negative APP, showed a positive response pattern in SG and SBG. Ceruloplasmin had a positive correlation with Obel grade, heart rate on animals from SGB and number of steps on horses submitted to starch overload (SG and SBG). Ceruloplasmin, α-1-antitrypsin and haptoglobin concentrations increased in SBG, suggesting an inflammatory response in animals of this group. Changes in clinical parameters were also more evident in the SBG, corroborating the protein fractionation findings.

Protein fractionation and shotgun proteomics analysis of enriched bacterial cultures shed new light on the enzymatically catalyzed degradation of acesulfame.

The removal of organic micropollutants in municipal wastewater treatment is an extensively studied field of research, but the underlying enzymatic processes have only been elucidated to a small extent so far. In order to shed more light on the enzymatic degradation of the artificial sweetener acesulfame (ACE) in this context, we enriched two bacterial taxa which were not yet described to be involved in the degradation of ACE, an unknown Chelatococcus species and Ensifer adhaerens, by incubating activated sludge in chemically defined media containing ACE as sole carbon source. Cell-free lysates were extracted, spiked with ACE and analyzed via target LC-MS/MS, demonstrating for the first time enzymatically catalyzed ACE degradation outside of living cells. Fractionation of the lysate via two-dimensional fast protein liquid chromatography (FPLC) succeeded in a partial separation of the enzymes catalyzing the initial transformation reaction of ACE from those catalyzing the further transformation pathway. Thereby, an accumulation of the intermediate transformation product acetoacetamide-n-sulfonic acid (ANSA) in the ACE-degrading fractions was achieved, providing first quantitative evidence that the cleavage of the sulfuric ester moiety of ACE is the initial transformation step. The metaproteome of the enrichments was analyzed in the FPLC fractions and in the unfractionated lysate, using shotgun proteomics via UHPLC-HRMS/MS and label-free quantification. The comparison of protein abundances in the FPLC fractions to the corresponding ACE degradation rates revealed a metallo-ß-lactamase fold metallo-hydrolase as most probable candidate for the enzyme catalyzing the initial transformation from ACE to ANSA. This enzyme was by far the most abundant of all detected proteins and amounted to a relative protein abundance of 91% in the most active fraction after the second fractionation step. Moreover, the analysis of the unfractionated lysate resulted in a list of further proteins possibly involved in the transformation of ACE, most striking a highly abundant amidase likely catalyzing the further transformation of ANSA, and an ABC transporter substrate-binding protein that may be involved in the uptake of ACE into the cell.

Developed rapid and simple RP-HPLC method for simultaneous separation and quantification of bovine milk protein fractions and their genetic variants.

The aim was to develop a reliable rapid reversed-phase high-performance liquid chromatography (RP-HPLC) method to simultaneously determine the main bovine milk protein fractions, including their genetic variants. Compared to the previous studies, our method is able to separate the main protein fractions within 20 min of total run time. The method validation consisted of testing repeatability, reproducibility linearity, repeatability, and accuracy. The procedure was developed using raw individual, bulk, and commercially available heat-treated cow milk samples. The RSD of peak areas ranged from 1.43 to 3.16% within analytical day and from 3.29 to 6.70% across analytical days. The method can be applied to investigate both raw and heat-treated milk samples.

Gel electrophoresis/electroelution sorting fractionator combined with filter-aided sample preparation for deep proteomic analysis.

Sample preparation and protein fractionation are important issues for proteomic studies. Protein extraction procedures strongly affect the performance of fractionation methods by provoking protein dispersion in several fractions. The most notable exception is the gel-based electrophoretic protein fractionation due to its resolution and effectiveness of sodium dodecyl sulfate as a solubilizing agent, while its main limitation lies in the poor recovery of the gel-trapped proteins. We created a fractionator device to separate complex mixture of proteins and peptides that is based on the continuous gel electrophoresis/electroelution sorting of these molecules. In an unsupervised process, complex mixtures of proteins or peptides are fractionated into the gel while separated fractions are simultaneously and sequentially electroeluted to the solution containing wells. The performance of the device was studied for protein fractionation in terms of reproducibility, protein recovery, and loading capacity. In a setup free of sodium dodecyl sulfate, complex peptide mixtures can also be fractionated. More than 11,700 proteins were identified in the whole-cell lysate of the CaSki cell line by using the fractionator combined with the filter-aided sample preparation method and mass spectrometry analysis. Fractionator-based proteome characterization increased 1.7-fold the number of identified proteins compared to the unfractionated sample analysis.

Evaluating the Protein Value of Fresh Tropical Forage Grasses and Forage Legumes Using In Vitro and Chemical Fractionation Methods.

The objectives of the present study were (1) to assess the adequacy of the in vitro and chemical methods to predict post-ruminal crude protein supply (PRCP) from fresh tropical forage, and (2) to identify PRCP supply predictors. Twenty-three fresh forage grasses and 15 forage legumes commonly used in domestic cattle feeding in the tropics and subtropics were incubated in the rumen of cows to determine ruminal crude protein (CP) degradation. The PRCP supply was calculated from in situ rumen-undegraded CP and in vitro organic matter digestibility (i.e., reference method), from ammonia-nitrogen release during in vitro incubation (i.e., in vitro method), and from the concentrations of chemical CP fractions (i.e., chemical method). The adequacy was evaluated using error-index and dimensionless parameters, and stepwise regression was used to select PRCP predictors. Adequacy ranged from poor to moderate (0.53 to 0.74) for the in vitro method being lower for forage legumes at a slow rumen passage rate (0.20), and even poorer (0.02 to 0.13) for the chemical method. Hence, the in vitro method can estimate PRCP supply in tropical forages with moderate to high but not with slow passage rates. Equations developed in the present study appear to predict PRCP supply with reasonable adequacy.

Milk microfiltration process dataset annotated from a collection of scientific papers.

Milk microfiltration process plays a key role in the dairy industry. Crossflow microfiltration of skimmed milk using a membrane with 0.1 µm mean pore size is widely used to fractionate the two main groups of dairy proteins: casein micelles (~150 nm) and serum proteins (~2-15 nm). Retentate, containing mainly casein micelles, is generally used to enrich vat milk for cheese making. Permeate, containing serum proteins, lactose and minerals, is usually ultrafiltered in order to produce protein-rich concentrate with a high nutritional value dedicated to specific populations such as infants and seniors. The great interest in these protein fractions explains the increasing number of microfiltration equipments in the dairy industry. This data article contains data associated with milk microfiltration process experiments and properties of the resulting dairy fractions annotated from a collection of scientific documents. These data are stored in INRAE public repository (see Data accessibility in the Specification Table for direct links to data). They have been structured using MILK MICROFILTRATION ontology and are replicated in @Web data warehouse providing additional querying tools (https://www6.inrae.fr/cati-icat-atweb/).

Chromatography-Independent Fractionation and Newly Identified Molecular Features of the Adzuki Bean (Vigna angularis Willd.) ß-vignin Protein.

Adzuki seed ß-vignin, a vicilin-like globulin, has proven to exert various health-promoting biological activities, notably in cardiovascular health. A simple scalable enrichment procedure of this protein for further nutritional and functional studies is crucial. In this study, a simplified chromatography-independent protein fractionation procedure has been optimized and described. The electrophoretic analysis showed a high degree of homogeneity of ß-vignin isolate. Furthermore, the molecular features of the purified protein were investigated. The adzuki bean ß-vignin was found to have a native size of 146 kDa, and the molecular weight determined was consistent with a trimeric structure. These were identified in two main polypeptide chains (masses of 56-54 kDa) that are glycosylated polypeptides with metal binding capacity, and one minor polypeptide chain with a mass 37 kDa, wherein these features are absent. The in vitro analysis showed a high degree of digestibility of the protein (92%) and potential anti-inflammatory capacity. The results lay the basis not only for further investigation of the health-promoting properties of the adzuki bean ß-vignin protein, but also for a possible application as nutraceutical molecule.

Construction of a microfluidic platform integrating online protein fractionation, denaturation, digestion, and peptide enrichment.

Microfluidic system with multi-functional integration of high-throughput protein/peptide separation ability has great potential for improving the identification capacity of biological samples in proteomics. In this paper, a sample treatment platform was constructed by integrating reversed phase chromatography, immobilized enzyme reactor (IMER) and imprinted monolith through a microfluidic chip to achieve the online proteins fractionation, denaturation, digestion and peptides enrichment. We firstly synthesized a poly-allyl phenoxyacetate (AP) monolith and a lysine-glycine-glycine (KGG) imprinted monolith separately, and investigated in detail their performance in fractionating proteins and extracting KGG from the protein digests of MCF-7 cell. The removal percentage of 94.6% for MCF-7 cell protein and the recovery of 90.8% for KGG were obtained. The number of proteins and peptides identified on this microfluidic platform was 2,004 and 8,797, respectively, which was 2.8-fold and 3.0-fold higher than that of untreatment sample. The time consumed by this platform for a sample treatment was about 9.6 h, less than that of conventional method (approximate 13.3 h). In addition, this platform can enrich some peptide fragments containing KGG based on imprinted monolith, which can be served for the identification of ubiquitin-modified proteomics. The successful construction of this integrated microfluidic platform provides a considerable and efficient technical tool for simultaneous identification of proteomics and post-translational modification proteomics information.

A simple serum depletion method for proteomics analysis.

Serum is the body fluid most often used in biomarker discovery. Albumin, the most abundant serum protein, contributes approximately 50% of the serum protein content, with an additional dozen abundant proteins dominating the rest of the serum proteome. To profile this challenging protein mixture by proteomics, the abundant proteins must be depleted to allow for detection of the low-abundant proteins, the primary biomarker targets. Current serum depletion approaches for proteomics are costly and relatively complex to couple with protein digestion. We demonstrate a simple, affordable serum depletion methodology that, within a few minutes of processing, results in two captured serum fractions - albumin-depleted and albumin-rich - which are digested in situ. We believe our method is a useful addition to the biomarker sample preparation toolbox.

The Potential of Fractionated Rice Bran Protein Hydrolysates as Antioxidative and Anti-Inflammatory Agents.

Rice bran is generally used as animal feed despite containing numerous nutritional compounds. Small peptides possessing high antioxidant activity can be obtained from rice bran protein via enzymatic hydrolysis. Immune-modulating and antioxidative activity of rice bran protein hydrolysates from crude rice bran protein and its fractions were studied. Albumin, globulin, glutelin, and prolamin proteins were fractionated based on solubility differences and hydrolyzed with two types of enzyme, namely pepsin and protease M. Albumin fraction showed a high degree of hydrolysis in both enzymes. Protease M differently digested rice bran protein fractions, in which it showed low digestion in glutelin and prolamin fractions. After 30 min of hydrolysis time, the reaction slowed down, and antioxidant activity remained constant in pepsin hydrolysis. Due to the high presence of lipopolysaccharide (LPS) in protease M digested fractions (caused by the enzyme), it could not be used to determine immune-modulating activity. THP-1 macrophages were simultaneously stimulated with 100 ng/mL LPS and rice bran protein hydrolysates from 4 h of pepsin digestion. Reduction of pro-inflammatory cytokine IL-1ß and increase of anti-inflammatory cytokine IL-10 were observed from crude rice bran protein and albumin. In conclusion, pepsin-digested rice bran protein could be potentially used as antioxidative and anti-inflammatory agent.

Profiling of hydroxyurea-treated ß-thalassemia/ serum proteome through nano-LC-ESI-MS/ MS in combination with microsol-isoelectric focusing.

Advancements in proteomic tools offer a comprehensive solution to studying the complexity of diseases at molecular level. This study focusses on the clinical proteomic profiling of pre- and post-hydroxyurea (HU)-treated ß-thalassemia patients in parallel with healthy individuals to better understand the role of HU in the treatment of ß-thalassemia. The strategy encompasses sequential high-resolution protein fractionation using MicroSol-isoelectric focusing (ZOOM- IEF) followed by one-dimensional SDS-PAGE before nano-RP-LC-MS/ MS analysis of tryptic peptides. Protein identification was performed through Mascot search using NCBInr and SwissProt databases. Several different proteins were observed in pool serum samples of each of the three study groups. Approximately, 1250 proteins exclusive to each group were identified, and after removing the redundant and low sequence coverage proteins, the number was reduced to 576 (201 in healthy, 187 in HU-untreated and 188 in HU-treated group). Uniquely identified proteins in the HU-treated group regulate the focal adhesion, ECM-receptor interaction, PI3K-Akt signaling, Rap1 signaling, cAMP signaling, platelet activation, and Ca2+ signaling pathways in the HU-treated group. The proteomic profile presented here will add to the current state of understanding of molecular mechanisms involved in hydroxyurea treatment of ß-thalassemia.

Variations in Amino Acid and Protein Profiles in White versus Brown Teff (Eragrostis Tef) Seeds, and Effect of Extraction Methods on Protein Yields.

Data on variations in amino acid compositions and protein profiles among white and brown teff, a grain of growing interest, is either limited or contradicting at the moment. In this study, three white (Addis-W, Mekel-W and Debre-W) and three brown (Addis-B, Mekel-B and Debre-B) teff seed samples were used for whole flour amino acid analysis and protein fractionation with three different methods. White and brown seed types showed different physical changes during protein extraction. Brown teff displayed higher essential amino acid content than white with lysine present in high concentration in both seed types. Extraction with tert-butanol increased prolamin yields in teff compared to ethanol. The major protein fraction in teff was glutelin with white teff containing higher glutelin proportion than brown. Sodium Dodecyl Sulfate Gel Electrophoresis (SDS-PAGE) analysis revealed clear genetic variability between white and brown teff seed types.