Search

Olive cake, the solid byproduct of three-phase centrifugation olive oil production, has a high organic and polyphenol content, rendering it an environmental threat when landfilled as well as limiting its animal feed potential. This residue can be a good candidate for biomethane production due to its rich polysaccharide content (pectin, hemicellulose, and cellulose). Two strategies were compared to maximize biomethane production: destoning (i.e., removal of the seed fragments via mechanical means) and enzymatic pretreatment of the pulp. After 30 days of batch anaerobic digestion at 35 °C, both enzymatically pretreated and destoned olive cakes produced similar amounts of methane (~295 mL CH4/g volatile solids (VS)), 42% more than the control. A comparison of olive cake's biomethane yields with a broad range of agricultural residues in the literature demonstrated its suitability for biomethane production. Additionally, the digestate recovered from the anaerobic digestion of olive cake had high Kjeldahl nitrogen contents (3.6%, db) and low polyphenol concentrations (0.02 mg gallic acid equivalent (GAE)/g), qualifying it as an ingredient for soil amendment. This study demonstrated olive cake can be diverted from landfills for second-generation biofuel production, and that the resulting digestate may have value for soil amendment. [ABSTRACT FROM AUTHOR]

Chronic infection with the hepatitis B virus (HBV) affects an estimated 257 million people worldwide and can lead to liver diseases such as cirrhosis and liver cancer. Viral replication is generally considered not to be cytopathic, and although some HBV proteins may have direct carcinogenic effects, the majority of HBV infection-related disease is related to chronic inflammation resulting from disrupted antiviral responses and aberrant innate immune reactions. Like all cells, healthy and HBV-infected cells communicate with each other, as well as with other cell types, such as innate and adaptive immune cells. They do so by both interacting directly and by secreting factors into their environment. Such factors may be small molecules, such as metabolites, single viral proteins or host proteins, but can also be more complex, such as virions, protein complexes, and extracellular vesicles. The latter are small, membrane-enclosed vesicles that are exchanged between cells, and have recently gained a lot of attention for their potential to mediate complex communication and their potential for therapeutic repurposing. Here, we review how HBV infection affects the communication between HBV-infected cells and cells in their environment. We discuss the impact of these interactions on viral persistence in chronic infection, as well as their relation to HBV infection-related pathology. [ABSTRACT FROM AUTHOR]

Carrot processors produce approximately 175,000 tons of waste annually in the United State of America. Carrot waste conversion is important to the carrot processing industry as this waste is rich in bioactive compounds and dietary fiber. We evaluated the effects of hydraulic press and expeller press on liquid and bioactive compound extractions. Mechanical separation of carrot mash by expeller pressing improved liquid extraction over the hydraulic press while simultaneously increasing the total solid, carotenoid, and polyphenol contents. Compared to untreated control mash, mechanically treated mash had higher fat-binding capacity. Our study indicates that further conversion of carrot mash could lead to better value streams for this byproduct. [ABSTRACT FROM AUTHOR]

Recognition of RNA by receptors of the innate immune system is regulated by various posttranslational modifications. Different single 2′-O-ribose (2′-O-) methylations have been shown to convert TLR7/TLR8 ligands into specific TLR8 ligands, so we investigated whether the position of 2′-O-methylation is crucial for its function. To this end, we designed different 2′-O-methylated RNA oligoribonucleotides (ORN), investigating their immune activity in various cell systems and analyzing degradation under RNase T2 treatment. We found that the 18S rRNA-derived TLR7/8 ligand, RNA63, was differentially digested as a result of 2′-O-methylation, leading to variations in TLR8 and TLR7 inhibition. The suitability of certain 2′-O-methylated RNA63 derivatives as TLR8 agonists was further demonstrated by the fact that other RNA sequences were only weak TLR8 agonists. We were thus able to identify specific 2′-O-methylated RNA derivatives as optimal TLR8 ligands. [ABSTRACT FROM AUTHOR]

Objectives: Platelet activation underpins thrombus formation in ischemic stroke. The active, dimeric form of platelet receptor glycoprotein (GP) VI plays key roles by binding platelet ligands collagen and fibrin, leading to platelet activation. We investigated whether patients presenting with stroke expressed more GPVI on their platelet surface and had more active circulating platelets as measured by platelet P-selectin exposure. Methods: 129 ischemic or hemorrhagic stroke patients were recruited within 8h of symptom onset. Whole blood was analyzed for platelet-surface expression of total GPVI, GPVI-dimer, and P-selectin by flow cytometry at admission and day-90 post-stroke. Results were compared against a healthy control population (n = 301). Results: The platelets of stroke patients expressed significantly higher total GPVI and GPVI-dimer (P<0.0001) as well as demonstrating higher resting P-selectin exposure (P<0.0001), a measure of platelet activity, compared to the control group, suggesting increased circulating platelet activation. GPVI-dimer expression was strongly correlated circulating platelet activation [r2 = 0.88, P<0.0001] in stroke patients. Furthermore, higher platelet surface GPVI expression was associated with increased stroke severity at admission. At day-90 post-stroke, GPVI-dimer expression and was further raised compared to the level at admission (P<0.0001) despite anti-thrombotic therapy. All ischemic stroke subtypes and hemorrhagic strokes expressed significantly higher GPVI-dimer compared to controls (P<0.0001). Conclusions: Stroke patients express more GPVI-dimer on their platelet surface at presentation, lasting at least until day-90 post-stroke. Small molecule GPVI-dimer inhibitors are currently in development and the results of this study validate that GPVI-dimer as an anti-thrombotic target in ischemic stroke. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral components, mediating immune responses and likely the spread of the virus. However, the obstacles involved in purifying EVs and removing contaminating viral particles in a reliable and effective manner bottlenecks the full potential for the development of clinical and diagnostic treatment options targeting EV. Because of the similarities in size, density, membrane composition and mode of biogenesis of EVs and virions there are no standardized approaches for virus‐removal from EV preparations yet. Functional EV studies also require EV samples that are devoid of antibody contaminants. Consequently, the study of EVs in virology needs reliable and effective protocols to purify EVs and remove contaminating antibodies and viral particles. Here, we established a protocol for EV purification from hepatitis B virus (HBV)‐containing plasma by a combination of size‐exclusion chromatography and affinity‐based purification. After purification, EV samples were free of virus‐sized particles, HBV surface antigen, HBV core antigen, antibodies or infectious material. Viral genomic contamination was also decreased following purification. By using appropriate antibodies and size parameters, this protocol could potentially be applied to purification of EVs from other viral samples. In summary, we established a fast, reproducible and robust approach for the removal of HBV from EV preparations. Looking forward to the point of purifying EVs from clinical samples, this method should enable studies shedding light on the underlying mechanisms of EVs in viral infections and their diagnostic and prognostic potential. [ABSTRACT FROM AUTHOR]

Corn distillers dried grains with solubles (DDGS) are not nutritionally complete as a nonruminant ingredient owing to poor essential amino acid profile, and high fat and fiber contents. Coproducts of soybean enzyme‐assisted aqueous extraction process, skim (wastewater) and insoluble fiber (IF; solid residue), and/or enzymes (pectinase, cellulase, and acid protease; referred to as PCF) were evaluated as distillers dried grains (DDG) nutritional quality enhancers in corn fermentation. Corn‐soy DDG had ~10% higher protein, ~3% lower fat, and ~2% lower fiber contents compared to corn DDG; fiber content was further reduced with PCF treatment (~4% total decrease). Concentrations of all essential amino acids in corn‐soy DDG showed at least a threefold increase, except for allo‐isoleucine and tryptophan, compared to corn DDG. Corn‐soy DDG had ~25% decrease in total fatty acid (TFA) and ~6% decrease in free fatty acid (FFA) contents compared to corn DDG; TFA and FFA contents further decreased with PCF treatment. Corn‐soy DDG had ~15%, 3%, and 1.7% lower hemicellulose, cellulose, and lignin contents, respectively, compared to corn DDG; hemicellulose content further decreased with PCF treatment. Mineral composition of corn‐soy DDG was in the recommended range, except Na and S were out of range by 0.79% and 0.74%, respectively. All results, except for Na and S, suggest strong potential of using skim and IF as DDG nutritional quality enhancers. [ABSTRACT FROM AUTHOR]

Aqueous oil extraction is an approach that could replace organic solvent extraction with water. Compared to typical solvent extraction and mechanical pressing processes, aqueous extraction has higher oil recovery (over 80%) than the mechanical pressing process, and resolve issues resulted from chemical loading and remaining in the hexane extraction. Proteases are used to assist free oil release from oil bodies by hydrolyzing cotyledon cell walls in aqueous extraction process. The resulting enzyme-assisted aqueous extraction process (EAEP) includes dehulling, flaking, extruding, enzymatic extraction, and enzymatic demulsification processes. SuperPro Designer was used to conduct a techno-economic analysis (TEA) of the extraction process. The total capital investment, operation cost, and profits were evaluated. During EAEP, insolubility of water and oil allows the simultaneous extraction of protein and oil. This decreases operation costs, especially the oil purification process, and therefore increases profits made from the main product (soybean oil). This simultaneous extraction also increases the profit towards the coproduct, i.e., protein in skim. Additionally, the absence of chemical and enzyme recycling contribute to the better economic value of EAEP. Despite the increase in facility costs due to extraction and demulsification units, the value-added coproduct extraction and high free oil yield contribute to the economic feasibility of EAEP in industrial- and commercial-scale productions when skim and insoluble fiber are used as water and carbohydrate supplies for integrated soy/corn ethanol biorefinery processing. [ABSTRACT FROM AUTHOR]

Insoluble fiber (IF) recovered from the enzyme-assisted aqueous extraction process (EAEP) of soybeans is a fraction rich in carbohydrates and proteins. It can be used to enhance ethanol production in an integrated corn-soy biorefinery, which combines EAEPwith traditional corn-based ethanol processing. The present study evaluated IF as a substrate for ethanol production. The effects of treatment of IF (soaking in aqueous ammonia (SAA), liquid hot water (LHW), and enzymatic hydrolysis), primarily simultaneous saccharification and co-fermentation (SSCF), as well as scaling up (250 mL to 60 L) on ethanol production from IF alone or a corn and IF slurry were investigated. Enzymatic hydrolysis (pectinase, cellulase, and xylanase, each added at 5% soy solids during simultaneous saccharification and fermentation/SSCF) was the best treatment to maximize ethanol production from IF. Ethanol yield almost doubled when SSCF of IF was performed with Saccharomyces cerevisiae and Escherichia coli KO11. Addition of IF in dry-grind corn fermentation increased the ethanol production rate (~31%), but low ethanol tolerance of E. coli KO11 was a limiting factor for employing SSCF in combination corn and IF fermentation. Nonlinear Monod modeling accurately predicted the effect of ethanol concentration on E. coli KO11 growth kinetics by Hanes-Woolf linearization. Collectively, the results from this study suggest a potential of IF as a substrate, alone or in dry-grind corn fermentation, where it enhances the ethanol production rate. IF can be incorporated in the current bioethanol industry with no added capital investment, except enzymes. [ABSTRACT FROM AUTHOR]

Background: Platelets are essential to physiological hemostasis or pathological thrombus formation. Current antiplatelet agents inhibit platelet aggregation but leave patients at risk of systemic side-effects such as hemorrhage. Newer therapeutic strategies could involve targeting this cascade earlier during platelet adhesion or activation via inhibitory effects on specific glycoproteins, the thrombogenic collagen receptors found on the platelet surface. Aims: Glycoprotein VI (GPVI) is increasingly being recognized as the main platelet-collagen receptor involved in arterial thrombosis. This review summarizes the crucial role GPVI plays in ischemic stroke as well as the current strategies used to attempt to inhibit its activity. Summary of review: In this review, we discuss the normal hemostatic process, and the role GPVI plays at sites of atherosclerotic plaque rupture.We discuss how the unique structure of GPVI allows for its interaction with collagen and creates downstream signaling that leads to thrombus formation. We summarize the current strategies used to inhibit GPVI activity and how this could translate to a clinically viable entity that may compete with current antiplatelet therapy. Conclusion: From animal models, it is clear that GPVI inhibition leads to an abolished platelet response to collagen and reduced platelet aggregation, culminating in smaller arterial thrombi. There is now an increasing body of evidence that these findings can be translated into the development of a bleeding free pharmacological entity specific to sites of plaque rupture in humans. [ABSTRACT FROM AUTHOR]

TLR7 and TLR8 recognize RNA from pathogens and lead to subsequent immune stimulation. Here we demonstrate that a single naturally occurring 2’-O-methylation within a synthetic 18s rRNA derived RNA sequence prevents IFN-α production, however secretion of proinflammatory cytokines such as IL-6 is not impaired. By analysing TLR-deficient plasmacytoid dendritic cells and performing HEK293 genetic complementation assays we could demonstrate that the single 2’-O-methylation containing RNA still activated TLR8 but not TLR7. Therefore this specific 2’-O-ribose methylation in rRNA converts a TLR7 / TLR8 ligand to an exclusively TLR8-specific ligand. Interestingly, other modifications at this position such as 2’-O-deoxy or 2’-fluoro had no strong modulating effect on TLR7 or TLR8 activation suggesting an important role of 2’-O-methylation for shaping differential TLR7 or TLR8 activation. [ABSTRACT FROM AUTHOR]

A sublethally injured bacterial cell has been defined as a cell that survives a stress such as heating, freezing, acid treatment, or other antimicrobial intervention but can repair the cellular damage exerted by the stressor and later regain its original ability to grow. Consequently, sublethally injured cells are not likely to be included in conventional enumeration procedures, which could result in unrealistically low counts unless efforts are made to encourage recovery of the injured cells before enumeration. The objective of this study was to evaluate the use of the thin agar layer (TAL) method for the recovery of pressure-injured and heat-injured Listeria monocytogenes in a tryptic soy broth with 0.6% yeast extract system. Pressure injury consisted of treatment of a culture of mixed L. monocytogenes strains with high hydrostatic pressure at 400 or 600 MPa for 1 s, 2 min, 4 min, or 6 min at a process temperature of 12 ± 2°C. Heat injury consisted of treatment of a culture of mixed L. monocytogenes strains at 60 ± 1°C for 3, 6, or 9 min. Growth media were tryptic soy agar (TSA) with 0.6% yeast extract, modified Oxford medium (MOX), and TAL, which consisted of a 7-ml layer of TSA overlaid onto solidified MOX. Counts of viable L. monocytogenes on TAL were higher than those on MOX in the heat-injury experiment but not in the pressure-injury experiment. Therefore, the effectiveness of the TAL method may be specific to the type of injury applied to the microorganism and should be investigated in a variety of cellular injury scenarios. [ABSTRACT FROM AUTHOR]

Sodium nitrite exerts an inhibitory effect on the growth of Listeria monocytogenes. The objective of this study was to investigate the effects of various nitrite concentrations from a vegetable source with and without high hydrostatic pressure (HHP) on the recovery and growth of L. monocytogenes on ready-to-eat restructured ham. A preconverted celery powder was used as the vegetable source of nitrite. Targeted concentrations of natural nitrite investigated were 0, 50, and 100 mg/kg. HHP treatments evaluated were 400 MPa for 4 min and 600 MPa for 1 or 4 min at 12 ± 2°C (initial temperature of the pressurization fluid). Viable L. monocytogenes populations were monitored on modified Oxford medium and thin agar layer medium through 98 days of storage at 4 ± 1°C. Populations on both media did not differ. The HHP treatment at 600 MPa for 4 min resulted in L. monocytogenes populations below the detection limit of our sampling protocols throughout the storage period regardless of the natural nitrite concentration. The combination of HHP at 400 MPa for 4 min or 600 MPa for 1 min with natural nitrite resulted in initial inhibition of viable L. monocytogenes. Ham formulations that did not contain natural nitrite allowed faster growth of L. monocytogenes than did those with nitrite, regardless of whether they were treated with HHP. The results indicate that nitrite from a vegetable source at the concentrations used in this study resulted in slower growth of this microorganism. HHP treatments enhanced the inhibitory effects of natural nitrite on L. monocytogenes growth. Thus, the combination of natural nitrite plus HHP appears to have a synergistic inhibitory effect on L. monocytogenes growth. [ABSTRACT FROM AUTHOR]

BACKGROUND: Egg phosvitin could be a good source of functional peptides. Enzymatic dephosphorylation and high-pressure processing combined with thermal treatment applied before proteolysis could produce phosvitin hydrolysates with different properties compared to its native form. RESULTS: Phosvitin structure was maintained overall during high-pressure treatment of 600 MPa applied at an initial temperature of 65 °C regardless of the pH and duration of treatment, confirming the high structural stability of this phosphoprotein. Treatment of phosvitin with phosphatase increased the degree of dephosphorylation from 24% to 63%, after 2 and 18 h, respectively. Moderate dephosphorylation of phosvitin prior to proteolytic digestion improved its hydrolysis, allowing formation of peptides with a molecular weight lower than 17,000 kDa as determined by size exclusion chromatography. Angiotensin-converting enzyme (ACE) inhibition and antioxidant activity of dephosphorylated and protease-treated phosvitin was increased by 52% and 39%, respectively, as compared to protease-digested native phosvitin. CONCLUSION: Enzymatic dephosphorylation before proteolysis mimicking in vivo gut conditions improved ACE inhibition and antioxidant activity of phosvitin hydrolysates. Copyright © 2012 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

The effectiveness of soaking in aqueous ammonia (SAA) as a pretreatment method for the conversion of soybean fiber to ethanol via simultaneous saccharification and fermentation (SSF) was investigated. Insoluble fiber is a co-product from oil and protein extraction using two-stage, countercurrent, enzyme-assisted, aqueous extraction processing of full-fat soybean flakes (FFSF) and extruded FFSF. The fiber fractions were soaked in 15 wt% aqueous ammonia at 1:10 solid-to-liquid ratio. The effects of operating variables, including treatment times (6, 12, and 24 h), treatment temperatures (60 and 80 °C), and cellulase loadings (15 and 60 FPU/g-glucan) on the degree of enzymatic hydrolysis were determined. The best SAA conditions were 80 °C for 12 h followed by an enzyme loading of 15 FPU/g-glucan, which produced a 152-mg/g glucose yield after 48 h of hydrolysis. This was 8.7 times the amount produced from the same fiber not pretreated with SAA. The glucose yield increased to 381 mg/g when fiber obtained from extruded FFSF was submitted to SAA. SAA (80 °C, 12 h) on extruded fiber subjected to SSF increased ethanol yield from 0.06 g of ethanol/g [40% of theoretical yield] (for non SAA pretreated fiber) to 0.25 g of ethanol/g [92% of theoretical yield]. The combination of extrusion and SAA was an efficient means for converting the fiber-rich soybean fraction into ethanol. [ABSTRACT FROM AUTHOR]

Platelets in flowing blood at high-shear stress are recruited to exposed subendothelial collagen of injured vessels by GPIb–von Willebrand factor (vWf) and integrin α2β1 (α2β1)–collagen interactions. Platelet adhesion to type I collagen depends mainly on the α2β1–collagen interaction and that to type III collagen depends on the GPIb–vWf interaction due to vWf's weak affinity for type I collagen. Contributions of these two interactions would differ depending on expressions of α2β1, vWf, or GPIb. We quantitated platelet adhesion to low- and high-density collagen under high-shear flow conditions in the presence of anti-α2β1 (Gi9) and anti-GPIb (NNKY5-5) antibodies to determine if their inhibitory effects were correlated with the amounts of α2β1, GPIb and vWf. Gi9 inhibition of adhesion to type I collagen was decreased in platelets with more integrin α2β1. Gi9 and NNKY5-5 are more inhibitory against adhesion to low-density type III and I, respectively. Higher α2β1 expression decreases adhesion to low-density type III and increases Gi9 inhibition of adhesion to high-density type III, suggesting crosstalk between the α2β1–collagen and GPIb–vWf interactions in adhesion to type III. Integrin α2β1–collagen and GPIb–vWf interactions both contribute to platelet adhesion to collagen under high-shear flow. In adhesion under high-shear stress, the two interactions would compensate for each other, when there is a deficiency in one or the other. The α2β1–collagen interaction was also suggested to have an inhibitory effect on platelet adhesion to type III collagen, through a yet undefined mechanism. [ABSTRACT FROM AUTHOR]

This study aimed to determine the effect of pretreating defatted soy flakes with ultrasound on soy protein isolate (SPI) yield and functionality. Defatted soy flakes dispersed into water (16%, w/w) were sonicated for 30, 60 and 120 s at ultrasonic amplitudes of 21 and 84 µmpp (peak to peak amplitude in µm), representing low and high power, respectively. The power densities were 0.30 and 2.56 W mL−1, respectively. The SPI yield increased by 13 and 34%, after sonication for 120 s at low and high power, respectively. The sonication of defatted soy flakes for 120 s at the higher power level improved the SPI solubility by 34% at pH 7.0, while decreasing emulsification and foaming capacities by 12 and 26%, respectively, when compared to control SPI. Rheological behavior of the SPI was also modified with significant loss in consistency coefficient due to sonication. Some of these results could be explained by the loss of the protein native state with increased sonication time and power. [ABSTRACT FROM AUTHOR]

The objectives of the present study were to determine how extrusion (barrel temperature of 100 °C) and high-pressure processing (HPP, 200 and 500 MPa, 15 min, 25 °C) of full-fat soybean flakes (FFSF) modified the structure of soybean cotyledon cells, the protein interactions and the in vitro protease accessibility. Cellular disruption of the cotyledon cells was only observed for extruded FFSF. Extrusion and HPP at 500 MPa favored formation of insoluble protein aggregates, in which oil was entrapped. High pressure size exclusion chromatography (HPSEC) and extraction methods using buffers containing SDS and 2-mercaptoethanol suggested that noncovalent interactions were the main forces in protein aggregate formation during HPP 500 MPa and extrusion. Intermolecular cross-linking by disulfide bonding was also involved in insoluble aggregates, but at a lesser extent than noncovalent interactions. Extrusion and HPP 500-MPa treatment enhanced the proteolytic attack, while treatment at 200 MPa had no impact. Drastic changes in the peptide profile of the extracted proteins were, however, only observed for the enzyme-treated 500-MPa FFSF. Optimal oil and protein extraction yields required cellular disruption of cotyledon cells and hydrolysis of protein aggregates, which were obtained with enzyme-assisted aqueous extraction of extruded FFSF. [ABSTRACT FROM AUTHOR]

MicroRNAs are important regulators of gene expression that control both physiological and pathological processes such as development and cancer. Although their mode of action has attracted great attention, the principles governing their expression and activity are only beginning to emerge. Recent studies have introduced a paradigm shift in our understanding of the microRNA biogenesis pathway, which was previously believed to be universal to all microRNAs. Maturation steps specific to individual microRNAs have been uncovered, and these offer a plethora of regulatory options after transcription with multiple proteins affecting microRNA processing efficiency. Here we review the recent advances in knowledge of the microRNA biosynthesis pathways and discuss their impact on post-transcriptional microRNA regulation during tumour development. [ABSTRACT FROM AUTHOR]

Approximately 70 million humans worldwide are affected by chronic hepatitis D, which rapidly leads to liver cirrhosis and hepatocellular carcinoma due to chronic inflammation. The triggers and consequences of this chronic inflammation, induced by co-infection with the hepatitis D virus (HDV) and the hepatitis B virus (HBV), are poorly understood. Using CRISPR technology, we characterized the recognition of HDV mono- and co-infection by intracellular innate immunity and determined its influence on the viral life cycle and effector T-cell responses using different HBV and HDV permissive hepatoma cell lines. We showed that HDV infection is detected by MDA5 and -after a lag phase -induces a profound type I interferon response in the infected cells. The type I interferon response, however, was not able to suppress HDV replication or spread, thus providing a persistent trigger. Using engineered T-cells directed against the envelope proteins commonly used by HBV and HDV, we found that HDV immune recognition enhanced T-cell cytotoxicity. Interestingly, the T-cell effector function was enhanced independently of antigen presentation. These findings help to explain immune mediated tissue damage in chronic hepatitis D patients and indicate that combining innate triggers with T-cell activating therapies might allow for a curative approach. [ABSTRACT FROM AUTHOR]

Over recent years, the food industry has striven to reduce waste, mostly because of rising awareness of the detrimental environmental impacts of food waste. While the edible oils market (mostly represented by soybean oil) is forecasted to reach 632 million tons by 2022, there is increasing interest to produce non-soybean, plant-based oils including, but not limited to, coconut, flaxseed and hemp seed. Expeller pressing and organic solvent extractions are common methods for oil extraction in the food industry. However, these two methods come with some concerns, such as lower yields for expeller pressing and environmental concerns for organic solvents. Meanwhile, supercritical CO2 and enzyme-assisted extractions are recognized as green alternatives, but their practicality and economic feasibility are questioned. Finding the right balance between oil extraction and phytochemical yields and environmental and economic impacts is challenging. This review explores the advantages and disadvantages of various extraction methods from an economic, environmental and practical standpoint. The novelty of this work is how it emphasizes the valorization of seed by-products, as well as the discussion on life cycle, environmental and techno-economic analyses of oil extraction methods. [ABSTRACT FROM AUTHOR]

RNAi is widely applied to inhibit expression of specific genes, but it is limited by variable efficiency and specificity of empirically designed sIRNA or shRNA constructs. This complicates studies targeting individual genes and significantly impairs large-scale screens using genome-wide knockdown libraries. Here, we show that ectopic expression of the RISC slicer Argonaute-2 (Ago2, eIF2C2) dramatically enhances RNA1 specifically for mRNA targets with perfectly matched binding sites. This effect depends on its endonuclease activity and is uncoupled from its regulation of microRNA expression. To model the application of Ago2 coexpression with shRNA knockdown, we targeted the EGF receptor (EGFR) in lung cancer cells exhibiting oncogene addiction to EGFR. Whereas multiple empirically designed shRNA constructs exhibited highly divergent efficiencies in mediating EGFR knockdown and cell killing, coexpression of Ago2 resulted in uniform and highly specific target gene suppression and apoptosis in EGFR-dependent cells. Codelivery of Ago2 with shRNA constructs or siRNA duplexes thus provides a strategy to enhance the efficacy and the specificity of RNAi in experimental and potentially therapeutic settings. [ABSTRACT FROM AUTHOR]

Of the two physiologically important platelet collagen receptors, glycoprotein (GP) VI is the receptor responsible for platelet activation. However, its reactivities towards different types of vascular collagen have not been directly and quantitatively analysed with collagen preparations of defined composition, although the other major platelet collagen receptor integrin α2β1 was shown to react with collagen types I-VI and VIII under either static or flow conditions. We analysed the collagen type specificity of GPVI binding to identify the physiological contribution of the various vascular collagens and how platelet reactivity towards the various collagens may be affected by fibril size. We used two methods to analyse the binding of recombinant GPVI (GPVI-Fc2) to different types of bovine collagen: binding to collagen microparticles in suspension and binding to immobilized collagen. GPVI-Fc2 bound to type I-III collagens that can form large fibrils, but not to type V that only forms small fibrils. The apparent GPVI binding to types IV and V could be ascribed to type I collagen that was a contaminant in each of these preparations. Kinetic analyses of the binding data showed that type III collagen fibrils have both a higher Kd and Bmax than types I and II. Flow adhesion studies demonstrated that type III collagen supports the formation of larger platelet aggregates than type I. Our present results suggest that the physiological importance of type III collagen is to induce thrombus formation. Furthermore, these studies indicate that GPVI mainly binds to collagen types that can form large collagen fibrils. [ABSTRACT FROM AUTHOR]

BSTRACT: The influence of soybean-to-water ratio (1:6 and 1:8) and pH (6 and 7) on characteristics of soymilk treated by high pressure processing (HPP, 500 and 600 MPa, 10 min, 25 °C) or thermal treatment (95 °C for 30 min) was investigated. Changes in the soymilk appearance were observed only for the 1:6, pH 6 soymilk that underwent a sol formation after HPP treatment and was apparently more viscous after thermal treatment. These changes were reflected in the viscosity values of these soymilks, which were increased by a factor of 4.3 and 3.6, after HPP and thermal treatment, respectively. After HPP treatment at pH 7 at both 1:6 and 1:8 ratios, the viscosity of the soymilks was unchanged compared to the controls. HPP treatment significantly increased the emulsion stability for all soymilks tested except for the 1:6, pH 7 soymilk. Peptide profiles of thermal- and pressure-treated samples were not affected; however, aggregates were observed in the soluble protein fraction after thermal and pressure treatment. Surface free and total free sulfhydryl content, surface hydrophobicity, and solubility of the proteins were decreased after HPP and thermal treatment regardless the soybean-to-water ratios and pH values of the soymilk. [ABSTRACT FROM AUTHOR]

Changes in the native state and functional properties of soy protein achieved by limited proteolysis of soy flour were investigated. Different enzyme-to-substrate ratios (E/S) were used to obtain low (3-5%) and medium (5-10%) degrees of hydrolysis (DH). Six protease preparations (three with predominately exopeptidase activities and three with predominately endopeptidase activities) were evaluated, and their effects on solubility, emulsification capacity, SDS-PAGE profiles, and denaturation enthalpies were characterized. Endoproteases (Multifect® Neutral, Protex™ 6L, and Multifect® P-3000) and exoproteases (Fungal Protease Concentrate, Experimental Fungal Protease #1, and Experimental Fungal Protease #2) yielded similar increases in soy protein solubility. The modifications to the soy peptide profile were similar for the three exoprotease mixtures at a 1% E/S ratio, whereas the extent of hydrolysis with Protex™ 6L was more pronounced than with the two other endoproteases (Multifect® Neutral and Multifect® P-3000). The emulsification capacity of protease-modified soy flour declined regardless of DH and enzyme type (exo- or endoprotease). After hydrolysis to >4% DH, denaturation enthalpies of glycinin and β-conglycinin decreased significantly, whereas hydrolysis to lower DH did not affect these values. [ABSTRACT FROM AUTHOR]

There is extensive evidence that FcR γ-chain couples to the collagen receptor glycoprotein VI (GPVI) and becomes phosphorylated on tyrosines upon receptor cross-linking. However, it is not established whether this receptor complex is sufficient to initiate the signalling cascade. We transfected GPVI and the FcR γ-chain into the human erythroleukaemia cell line K562, which lacks detectable expression of GPVI and the FcR γ-chain. The results show that GPVI is unable to signal when expressed alone, despite its surface expression, upon stimulation with the snake C-type lectin, convulxin. Coexpression of the FcR γ-chain confers signalling properties on the receptor. Furthermore, cotransfection of the FcR γ-chain and two mutant versions of GPVI shows that the transmembrane arginine and cytoplasmic tail of GPVI are necessary for association with the FcR γ-chain. These results demonstrate that reconstitution of the GPVI–FcR γ-chain complex in cells expressing the necessary signalling network is sufficient to initiate signalling events in response to convulxin and collagen-related peptide. [ABSTRACT FROM AUTHOR]

In agonist-induced platelet activation, the collagen platelet receptor integrin alpha[sub 2]beta[sub 1] is activated to high-affinity states through ADP involvement [Jung, S.M. & Moroi, M. (2000) J. Biol. Chem. 275, 8016 8026]. Here we determined the ADP-receptor subtypes involved and their relative contributions to alpha[sub 2]beta[sub 1] activation (assessed by soluble-collagen binding) using the P2Y12 antagonist AR-C69931MX and P2Y1 antagonists adenosine 3',5'-diphosphate (Ado(3,5)PP) and adenosine 3'-phosphate 5'-phosphosulfate (AdoPPS). All three inhibited alpha[sub 2]beta[sub 1] activation induced by low or high ADP, low thrombin, or low collagen-related peptide (CRP) concentrations; however, AR-C69931MX was markedly more inhibitory than the P2Y1 antagonists, suggesting the greater contribution of P2Y12. Inhibition patterns by various combinations of AR-C69931 MX, AdoPPS, and wortmannin suggested that P2Y1 and P2Y12 mediate alpha[sub 2]beta[sub 1] activation through different pathways, with possible involvement of phosphoinositide 3-kinase in both. Low concentrations of the acetoxy-methyl derivative of 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid (calcium chelator) markedly decreased o~2131 activation by low thrombin or CRP, but did not affect that by low or high ADP. Measurements of intracellular Ca[sup 2+] level (fluorimetric method) and alpha[sub 2]beta[sub 1] activation (soluble-collagen binding) in the same platelet preparation indicated that alpha[sub 2]beta[sub 1]activation via ADP receptors was independent of intracellular Ca[sup 2+] release. Our data indicate that integrin alpha[sup 2]beta[sub 1] activation by ADP occurs through an inside-out signaling mechanism involving differential contributions by P2Y1 and P2Y12 wherein oath contributes to some portion of the... [ABSTRACT FROM AUTHOR]

As a result of a viral infection, viral genomes are not only recognized by RIG-I, but also lead to the activation of RNase L, which cleaves cellular RNA to generate the endogenous RIG-I ligand (eRL). The eRL was previously identified as a specific sequence derived from the internal transcribed spacer region 2, which bears a 2′3′ cyclic phosphate instead of the common 5′ triphosphate. By now, the generation of the eRL and its immunostimulatory effect were shown both in vitro and in reporter systems. In this work, we aimed to elucidate whether the eRL is also generated in Influenza A (IAV) and vesicular stomatitis virus (VSV) infected cells. RNA was extracted from virus-infected cells and used for immunostimulations as well as specific PCR-strategies to detect eRL cleavage. We show that the eRL is generated in IAV infected HEK293 cells, but we could not detect specific eRL fragments in VSV infected cells. Further, RIG-I mediated IFN-response depends not only on viral genomes but also on the eRL, as immunostimulatory properties remain present under 5′triphosphate degrading conditions. In summary, we prove the IAV infection induced eRL generation in HEK293 cells, amplifying the innate immune response. [ABSTRACT FROM AUTHOR]

Citalopram, a selective serotonin reuptake inhibitor (SSRI), inhibits platelet function in vitro. We have previously shown that this action is independent of citalopram’s ability to block serotonin uptake by the serotonin transporter and must therefore be mediated via distinct pharmacological mechanisms. We now report evidence for two novel and putative mechanisms of citalopram-induced platelet inhibition. Firstly, in platelets, citalopram blocked U46619-induced Rap1 activation and subsequent platelet aggregation, but failed to inhibit U46619-induced increases in cytosolic Ca2+. Similarly, in neutrophils, citalopram inhibited Rap1 activation and downstream functions but failed to block PAF-induced Ca2+ mobilisation. In a cell-free system, citalopram also reduced CalDAG-GEFI-mediated nucleotide exchange on Rap1B. Secondly, the binding of anti-GPVI antibodies to resting platelets was inhibited by citalopram. Furthermore, citalopram-induced inhibition of GPVI-mediated platelet aggregation was instantaneous, reversible and displayed competitive characteristics, suggesting that these effects were not caused by a reduction in GPVI surface expression, but by simple competitive binding. In conclusion, we propose two novel, putative and distinct inhibitory mechanisms of action for citalopram: (1) inhibition of CalDAG-GEFI/Rap1 signalling, and (2) competitive antagonism of GPVI in platelets. These findings may aid in the development of novel inhibitors of CalDAG-GEFI/Rap1-dependent nucleotide exchange and novel GPVI antagonists. [ABSTRACT FROM AUTHOR]

Characterization and destabilization of the emulsion formed during aqueous extraction of oil from soybean flour were investigated. This emulsion was collected as a cream layer and was subjected to various single and combined treatments, including thermal treatments and enzymatic treatments, aimed at recovery of free oil. The soybean oil emulsion formed during the aqueous extraction processing of full fat flour contains high molecular weight glycinin and ß-conglycinin proteins and smaller oleosin proteins, which form a multilayer interface. Heat treatment alone did not modify the free oil recovery but freeze--thaw treatment increased the oil yield from 3 to 22%. After enzymatic treatment of the emulsion, its mean droplet size changed from 5 to 14 µm and the oil recovery increased to 23%. This increase could be attributed to the removal (due to enzymatic hydrolysis) of large molecular weight polypeptides from the emulsion interface, resulting in partial emulsion destabilization. When enzymatic treatment was followed by a freeze--thaw step, the oil recovery increased to 46%. This result can be attributed to the thinner interfacial membrane after enzymatic hydrolysis, partial coalescence during freeze--thaw, and coalescence during centrifugation. Despite the reduction in emulsion stability achieved, additional demulsification approaches need to be pursued to obtain an acceptably high conversion to free oil. [ABSTRACT FROM AUTHOR]

BSTRACT: Endo-protease treatments achieving low degrees of hydrolysis (DH 2% and 4%) were used to improve functional properties of hexane-extracted soy flour (HESF), extruded-expelled partially defatted soy flour (EESF), ethanol-washed soy protein concentrate (SPC), and soy protein isolate (SPI). These substrates had protein dispersibility indices ranging from 11% to 89%. Functional properties, including solubility profile (pH 3 to 7), emul-sification capacity and stability, foaming capacity and stability, and apparent viscosity were determined and related to surface hydrophobicity and peptide profiles of the hydrolysates. Protein solubilities of all substrates increased as DH increased. Emulsification capacity and hydrophobicity values of the enzyme-modified HESF and EESF decreased after hydrolysis, whereas these values increased for SPC and SPI. Emulsion stability was improved for all 4% DH hydrolysates. Hydrolyzed SPC had lower foaming capacity and stability. For substrates other than SPC, foaming properties were different depending on DH. Hydrolysis significantly decreased the apparent viscosities regardless of substrate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated differences in the molecular weight profiles of the hydrolysates. HESF and EESF, which had high proportions of native-state proteins, showed minor changes in the peptide profile due to hydrolysis compared with SPC and SPI. [ABSTRACT FROM AUTHOR]