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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]

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]

The disruption of chromatin structure can result in transcription initiation from cryptic promoters within gene bodies. While the passage of RNA polymerase II is a well-characterized chromatin-disrupting force, numerous factors, including histone chaperones, normally stabilize chromatin on transcribed genes, thereby repressing cryptic transcription. DNA replication, which employs a partially overlapping set of histone chaperones, is also inherently disruptive to chromatin, but a role for DNA replication in cryptic transcription has never been examined. In this study, we tested the hypothesis that, in the absence of chromatin-stabilizing factors, DNA replication can promote cryptic transcription in Saccharomyces cerevisiae. Using a novel fluorescent reporter assay, we show that multiple factors, including Asf1 , CAF-1, Rtt106 , Spt6 , and FACT, block transcription from a cryptic promoter, but are entirely or partially dispensable in G1-arrested cells, suggesting a requirement for DNA replication in chromatin disruption. Collectively, these results demonstrate that transcription fidelity is dependent on numerous factors that function to assemble chromatin on nascent DNA. [ABSTRACT FROM AUTHOR]

Commercial isolated yellow field pea protein isolate (IPP) was subjected to 200, 400 and 600 MPa high hydrostatic pressure (HHP) treatments followed by determination of some physicochemical and functional properties. Native polyacrylamide gel electrophoresis confirmed HHP-induced formation of high molecular weight protein aggregates. Intrinsic fluorescence showed most intense at 600 MPa where fluorescence intensity was less than half of the control IPP. The 600 MPa-treated IPP also showed more unfolded structure with an extensive red shift (384 nm) in wavelength of maximum tryptophan fluorescence. However, solubility profile was very similar and was not significantly ( p > 0.05) affected by HHP treatment. At pH 3.0, HHP-treated IPP formed significantly ( p < 0.05) higher quality emulsions with oil droplet sizes ( d 4,3 ) of 26–68 μm when compared to 52–92 μm for the control IPP. Foaming capacity was also higher at pH 3.0 with a maximum value of 81% when compared to maximum values of 38% and 62% obtained, respectively at pH 5.0 and 7.0. [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]

Enzyme-assisted aqueous extraction processing (EAEP) is an environmentally-friendly alternative to solvent and mechanical oil extraction methods, and can achieve ∼97% oil recovery from soybeans. The present study utilized soy skim (protein rich) and insoluble fiber (IF; carbohydrate rich), both co-products of EAEP, in dry-grind corn fermentation. The effects of adding soy skim and untreated IF (UIF), either separately or together, and adding pretreated IF (TIF), on ethanol production were investigated. Maximum ethanol production was achieved when UIF and skim were slurried together (corn-to-UIF ratio 1:0.16; skim-to-UIF ratio 6.5:1) and when fiber-hydrolyzing enzymes were added to corn fermentation. This modification to corn fermentation increased ethanol yield by 20%, ethanol production rate by 3%, and decreased fermentation time by 38 h compared to corn-only fermentation. An attempt was also made to utilize pentoses (from soy skim and IF) in integrated corn-soy fermentation slurry by an additional Escherichia coli KO11 fermentation step. [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]

Abstract: The effect of high pressure treatment on whole egg white was examined using in vitro pepsin digestion and proteomic methods. Pepsin incubations conducted with an enzyme to protein ratio of 3:1 following high pressure treatment (400–800MPa and 9°C) resulted in increased hydrolysis of egg white proteins. Pressure treatment of egg white at 800MPa resulted in greater susceptibility to pepsin hydrolysis than did thermal treatment at 95°C. The effect of 800MPa pressure treatment on egg white proteins was additionally examined by incubation with pepsin at an enzyme to protein ratio of 1:20 followed by 2-D electrophoresis. Results of these experiments showed extensive hydrolysis of most egg white proteins. Subsequent LC-MS/MS investigation of the low Mr fraction (<3.0kDa) derived from pepsin digested 800MPa treated egg white contained numerous peptides previously shown to have bioactive and/or immunological properties. Industrial Relevance: Short time high pressure treatment of whole egg white at relatively low temperature (9°C) resulted in increased pepsin digestibility equivalent to or better than heat treatment at 95°C. Examination of the peptides resulting from high pressure treatment and pepsin digestion revealed sequences with known biological activities. Thus high pressure treatment represents a promising technology for enhancing egg white''s healthiness and contributing to its role as a functional food. [Copyright &y& Elsevier]

The platelet collagen receptor glycoprotein VI (GPVI) has been suggested to function as a dimer, with increased affinity for collagen. Dissociation constants (Kd) obtained by measuring recombinant GPVI binding to collagenous substrates showed that GPVI dimers bind with high affinity to tandem GPO (Gly-Pro-Hyp) sequences in collagen, whereas the markedly lower affinity of the monomer for all substrates implies that it is not the collagen-binding form of GPVI. Dimer binding required a high density of immobilized triplehelical (GPO)10-containing peptide, suggesting that the dimer binds multiple, discrete peptide helices. Differential inhibition of dimer binding by dimer-specific antibodies, m-Fab-F and 204-11 Fab, suggests that m-Fab-F binds at the collagen-binding site of the dimer, and 204-11 Fab binds to a discrete site. Flow cytometric quantitation indicated that GPVI dimers account for ∼29% of total GPVI in resting platelets, whereas activation by either collagen-related peptide or thrombin increases the number of dimers to ∼39 and ∼44%, respectively. m-Fab-F inhibits both GPVI-dependent static platelet adhesion to collagen and thrombus formation on collagen under low and high shear, indicating that pre-existing dimeric GPVI is required for the initial interaction with collagen because affinity of the monomer is too low to support binding and that interaction through the dimer is essential for platelet activation. These GPVI dimers in resting circulating platelets will enable them to bind injury-exposed subendothelial collagen to initiate platelet activation. The GPVI-specific agonist collagen-related peptide or thrombin further increases the number of dimers, thereby providing a feedback mechanism for reinforcing binding to collagen and platelet activation. [ABSTRACT FROM AUTHOR]

Dynamic changes in mobile phase composition during high-performance liquid chromatography (HPLC) gradient elution coupled to mass spectrometry (MS) sensitively affect electrospray modes. We investigate the impact of the eluent composition on spray stability and MS response by infusion and injection experiments with a small tetrapeptide in water-acetonitrile mixtures. The employed HPLC/electrospray (ESI)-MS configuration uses a microchip equipped with an enrichment column, a separation column, and a makeup flow (MUF) channel. One nano pump is connected to the separation column, while a second one delivers solvent of exactly inverted composition to the MUF channel. Both solvent streams are united behind the separation column, before the ESI tip, such that the resulting electrosprayed solution always has identical composition during a gradient elution. Analyte peak parameters without and with MUF compensation are determined and discussed with respect to the electrospray mode and eluent composition. The postcolumn MUF significantly improves spray and signal stability over the entire solvent gradient, without compromising the performance of the HPLC separation column. It can also be conveniently implemented on microchip platforms. [ABSTRACT FROM AUTHOR]

Abstract: Changes in mobile phase composition during high performance liquid chromatography (HPLC) gradient elution coupled to mass spectrometry (MS) sensitively affect electrospray operation modes. In this work, we identify the influences of dynamic changes in bulk conductivity on the cone-jet stability island for aqueous acetonitrile and aqueous methanol mobile phases commonly used in reversed-phase HPLC. Bulk conductivities of the mobile phases were varied by adding different amounts of formic acid. A commercial microchip-HPLC/ESI-MS configuration was modified to enable in situ electrospray diagnostics by frequency analysis of the microchip emitter current and spray imaging. This approach facilitated the detection of different spray modes together with their onset potentials. The established spray modes are described and the differences in onset potentials and stability regions explained by the physicochemical properties of the electrosprayed liquid. [Copyright &y& Elsevier]

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]

Abstract: Interparticle void volumes and porosities of packed capillaries have been determined using intraparticle Donnan exclusion of a small, unretained, co-ionic tracer (nitrate ions). The operational domain of this approach has been characterized for bare silica, reversed-phase, and strong cation-exchange materials (with different particle sizes and intraparticle pore sizes) in dependence of the mobile phase ionic strength. Interparticle porosities agree well with those analyzed by inverse size-exclusion chromatography (ISEC). Limitations to the use of Donnan exclusion (electrostatic exclusion) and ISEC (mechanical exclusion) arise as either type of exclusion becomes noticeable also in the cusp regions between the particles, or as the intraparticle pores are so large that complete electrostatic and size-exclusion are difficult to realize. Our data confirm that intraparticle Donnan exclusion presents a most simple, fast, and reliable approach for the analysis of packing densities. [Copyright &y& Elsevier]

This work investigates the impact of conduit geometry on the chromatographic performance of typical particulate microchip packings. For this purpose, high-performance liquid chromatography (HPLC)/UV-microchips with separation channels of quadratic, trapezoidal, or Gaussian cross section were fabricated by direct laser ablation and lamination of multiple polyimide layers and then slurry- packed with either 3 or 5 μm spherical porous C8-silica particles under optimized packing conditions. Experimentally determined plate height curves for the empty microchannels are compared with dispersion coefficients from theoretical calculations. Packing densities and plate height curves for the various microchip packings are presented and conclusively explained. The 3 μm packings display a high packing density irrespective of their conduit geometries, and their performance reflects the dispersion behavior of the empty channels. Dispersion in 5 μm packings correlates with the achieved packing densities, which are limited by the number and accessibility of corners in a given conduit shape. [ABSTRACT FROM AUTHOR]

Abstract: Oil, protein and solid extraction yields obtained during aqueous extraction processing (AEP) of full fat soybean flakes (FFSF), FFSF extruded at a die temperature of 100°C and FFSF pressurised at 200 and 500MPa for 15min at 25°C, were compared to those obtained during enzyme-assisted aqueous extraction processing (EAEP) using 0.5% of protease Protex 7L. Without enzyme addition, pretreatment of the FFSF with extrusion and 500MPa increased and decreased, respectively, the oil extraction yield while protein extraction yield was significantly decreased after both treatments. The best treatment in terms of oil and protein recovery was EAEP of extruded flakes with 90% and 82% of oil and protein extraction yield, respectively, and 17% of free oil. Addition of protease during extraction significantly decreased the yield of isolated soy protein (ISP) due to an increased solubility of the proteins at pH 4.5. ISP from extruded EAEP had higher solubility at pH 7.0 and better functionality. The DSC results, combined with the protein extraction yields, showed that a proportion of the proteins became insoluble after extrusion and 500MPa treatment, while only those extracted from 500MPa FFSF had a reduced native state. [Copyright &y& Elsevier]

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]

Abstract: HPLC microchips are investigated experimentally with respect to packing density, pressure drop–flow rate relation, hydraulic permeability, and separation efficiency. The prototype microchips provide minimal dead volume, on-chip UV detection, and a 75mm long separation channel with a ca. 50μm×75μm trapezoidal cross-section. A custom-built stainless-steel holder allowed to adopt optimized packing conditions. Separation channels were slurry-packed with 3, 5, and 10μm-sized spherical, porous C8-silica particles. Differences in interparticle porosity, permeability, and plate height data are analyzed and consistently explained by different microchannel-to-particle size (particle-aspect) ratios and particle size distributions. [Copyright &y& Elsevier]

Abstract: High hydrostatic pressure was applied to hydrated soybeans (100–700MPa, 25°C) and soymilk (400–750MPa; 25 and 75°C) to assess its effect on isoflavone content, profile and water-extractability. Neither pressure level nor initial treatment temperature affected soymilk isoflavone content. However, combined pressure and mild thermal treatment modified the isoflavone distribution. At 75°C, the isoflavone profile shifted from malonylglucosides toward β-glucosides, which was correlated to the effect of adiabatic heating. When pressure was applied to the hydrated soybeans, the soymilk isoflavone concentration varied between 4.32 and 6.06μmol/g. The content of protein decreased and fat increased in soymilks prepared from pressurized soybeans with increasing pressure level. [Copyright &y& Elsevier]

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]

Abstract: The abundant Rap1 in platelets becomes activated when these cells are stimulated by various agonists, but its function has remained unknown. In view of this, we developed an assay to quantitatively measure activated Rap1 and used it to determine relationships between Rap1 activation and several platelet functions: integrin α2β1 activation, tyrosine phosphorylation, and the release reaction. We looked at how these processes are affected by the protein kinase C inhibitor BIMI, tyrosine kinase inhibitor PP2, PI 3-kinase inhibitor wortmannin, and ADP scavenger apyrase. In CRP (collagen related peptide)-activated platelets, all the inhibitors severely inhibited Rap1 activation, but had little effect on integrin α2β1 activation, indicating that the integrin activation mechanism is different from the Rap1 activation mechanism, at least in GPVI-dependent activation. With p85α-null mouse platelets, we demonstrated that Rap1 activation involves PI 3-kinase p85α-dependent tyrosine phosphorylation. All the inhibitors similarly decreased Rap1 activation and the serotonin release reaction, and the inhibition of Rap1 activation was not due to the lack of released ADP. Our results indicate that platelet Rap1 activation is closely related to the release reaction and not to integrin α2β1 activation in GPVI-mediated platelet activation. [Copyright &y& Elsevier]

Glycoprotein (GP) VI is a platelet membrane protein with a molecular weight of 62 kDa that was identified as a physiological collagen receptor from studies of patients deficient in this protein. GPVI-deficient platelets lacked specifically collagen-induced aggregation and the ability to form thrombi on a collagen surface under flow conditions, suggesting that GPVI makes an indispensable contribution to collagen-induced platelet activation. On the platelet surface, GPVI is present as a complex with the Fc receptor (FcR) γ-chain, probably composed of two GPVI molecules and one FcR γ-chain dimer. GPVI must form such a dimeric complex to exhibit high affinity binding to collagen. The GPVI-induced activation mechanism is initiated by tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of the FcR γ-chain, and then this signal is transduced to many related proteins, mainly by tyrosine phosphorylation. GPVI is widely recognized as a requisite factor for the formation of platelet aggregates on a collagen surface under blood flow. However, individuals with GPVI-deficient or null platelets do not exhibit any strong bleeding tendency. Analyzing this apparent dichotomy should provide us with a more precise understanding of the mechanism of thrombus formation. [Copyright &y& Elsevier]

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]

Integrated corn-soy fermentation, utilizing co-products of soybean enzyme-assisted aqueous extraction process (EAEP) of soybeans in corn fermentation, has shown potential for enhancing bioethanol production compared to corn only fermentation. To maximize economic returns, oil may be recovered. In the present study, the effect of skim and insoluble fiber, and oil extraction aids on ethanol yield, oil partition, and oil recovery, and quality of Distillers Dried Grains (DDG) was investigated. Two fiber hydrolyzing enzymes (pectinase and cellulase), an acid protease (Fermgen ® ), and a surfactant (Tween 80) were evaluated. Addition of skim, mixture of skim and insoluble fiber, or Fermgen ® to corn fermentation resulted in a ∼32 h decrease in fermentation time. Addition of soy co-products also resulted in ∼10–28% increase in oil partition in thin stillage with no additional enzyme or surfactant treatment. Addition of insoluble fiber alone resulted in ∼19% decrease in solids partition in thin stillage. Maximum free oil recovery, 22.5 ± 4.5%, was achieved from corn-insoluble fiber thin stillage with a combined treatment of enzymes (pectinase, cellulase, and Fermgen ® ) and surfactant (Tween 80). Maximum extractable oil recovery, 67 ± 3.2%, was achieved with the enzyme treatment alone. Corn-soy DDG has ∼11% higher protein, ∼2% lower fiber, and ∼2% lower fat contents compared to corn DDG. The fiber content was further reduced to ∼2% after enzyme treatment. This study demonstrates an efficient use of soy EAEP co-products and enzymes to maximize oil partition in thin stillage, and produce a high quality corn-soy DDG. [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]

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]

Commercial isolated pea protein dispersion (IPP, 1%, w/v) was pretreated with high pressure (200–600MPa, 5min at 24°C) or heat (100°C, 30min) prior to hydrolysis using 1–4% (w/w) alcalase concentrations. Fluorescence spectroscopy showed that heat pretreated IPP had a 35% higher level of exposed hydrophobic groups (measured as fluorescence intensity, FI) than the untreated protein. In contrast, the 200MPa pressure pretreatment produced a 15% increase in FI while 400 and 600MPa pretreatments, respectively, caused 5 and 60% decreases in FI. Heat pretreatment of IPP enhanced hydrolysis into smaller peptide sizes when compared to peptides from the 24°C pretreated protein. The 200MPa pretreatment enhanced IPP hydrolysis into smaller peptides, especially at lower (1–2%) alcalase concentrations. Protein hydrolysates produced from heat-pretreated IPP were less active against angiotensin converting enzyme (ACE) when compared to those from the 24°C pretreated protein. In general, heat or high pressure pretreatment of IPP favored production of ACE- and renin-inhibitory enhanced protein hydrolysates at a lower (1%) alcalase concentration. [ABSTRACT FROM AUTHOR]

[Display omitted] • EV sources tested for their applicability for EV bulk production. • sequential protocol for the mass purification of EVs from urine developed. • protocol is time-saving and cost-effective. • new methodology enables new therapeutic, diagnostic and technical developments. Research on extracellular vesicles (EVs), both in general laboratory methods and in novel therapeutic approaches, is limited by the difficulty of obtaining large quantities of EVs. This is due to the fact that EVs are only present at low concentrations in most starting samples and common methods for concentrating EVs from large volumes have decisive disadvantages. In particular, ultracentrifugation (UC) and tangential flow filtration (TFF) are used in EV bulk production. However, while UC leads to low EV-yield with high levels of impurities and damaged EVs, TFF is suitable for enriching EVs from large volumes, but requires expensive equipment. In addition, both methods require pre-purification of the EV material and downstream EV isolation steps. To circumvent all these obstacles, we evaluated various methods for their applicability to EV mass purification, optimized both their implementation and the workflow, and thus developed the following sequential protocol for EV bulk production: First, a preclearing step is performed by 1 µm filtration, followed by PEG precipitation to reduce total volume. Larger debris and apoptotic bodies are removed by centrifugation and filtration prior to enrichment and pre-purification of EVs by ultrafiltration or using the ExoEasyMaxi kit. Finally, EVs are purified by size exclusion chromatography. Since the availability of suitable starting material to obtain pure EVs is also a challenge, we compared different biological fluids, with urine proving to be the most suitable source. We determined the purity and quantity of the EV samples using nanotracking analysis, dynamic light scattering, phospholipid-, RNA- and protein-concentration measurement, electron microscopy and western blot. In summary, we have developed procedures to obtain about 1012 pure EVs in less than a day, with a handling time under 2 h, and at a cost of approximately €24. This method should facilitate further scientific and technical developments in the field of EV biology. [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]

Abstract: The effectiveness of several pretreatments [high-power ultrasound, sulfuric acid (H2SO4), sodium hydroxide (NaOH), and ammonium hydroxide (NH3OH)] to enhance glucose production from insoluble fractions recovered from enzyme-assisted aqueous extraction processing of extruded full-fat soybean flakes (FFSF) was investigated. Sonication of the insoluble fraction at 144μmpp (peak-to-peak) for 30 and 60s did not improve the saccharification yield. The solid fractions recovered after pretreatment with H2SO4 [1% (w/w), 90°C, 1.5h], NaOH [15% (w/w), 65°C, 17h], and NH3OH [15% (w/w), 65°C, 17h] showed significant lignin degradation, i.e., 81.9%, 71.2%, and 75.4%, respectively, when compared to the control (7.4%). NH3OH pretreatment resulted in the highest saccharification yield (63%) after 48h of enzymatic saccharification. A treatment combining the extraction and saccharification steps and applied directly to the extruded FFSF, where oil extraction yield and saccharification yield reached 98% and 43%, respectively, was identified. [Copyright &y& Elsevier]

Abstract: This research focused on the use of high-power ultrasound prior to soy protein extraction to simultaneously enhance protein and sugar release in the extract. Defatted soy flakes dispersed in water were sonicated for 15, 30, 60 and 120s using a bench-scale ultrasound unit. The ultrasonic amplitudes used were: 0, 21, 42, 63 and 84μmpp (peak-to-peak). The respective power densities were 0.30, 0.87, 1.53 and 2.56W/ml. Scanning electron micrographs of sonicated samples showed the structural disruption of soy flakes. The particle size decreased nearly 10-fold following ultrasonic treatment at high amplitudes. Sonication at high amplitude for 120s gave the highest increase in total sugar released (50%) and protein yield (46%) when compared with non-sonicated samples (control). Ultrasonic pretreatment was also carried out with and without cooling for temperature moderation. The heat generated during sonication had no significant effect on protein and sugar release from defatted soy flakes. The use of ultrasound can significantly improve protein yield and reduce the overall cost of producing soy protein from flakes. [Copyright &y& Elsevier]

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]

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]

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]

Abstract Background and aims Platelets play a fundamental role in the increased atherothrombotic risk related to central obesity since they show hyperactivation and lower sensitivity to antiplatelet therapy in obese patients. The main goal of this study was to identify platelet biomarkers related to the risk of atherothrombosis in obese patients, confirm platelet activation levels in these patients, and identify altered activation pathways. Methods Platelets were obtained from cohorts of obese patients and age- and sex-matched lean controls. Biochemical and proteome analyses were done by two-dimensional differential in-gel electrophoresis (2D-DIGE), mass spectrometry, and immunoblotting. Functional and mechanistic studies were conducted with aggregation assays and flow cytometry. Results We confirmed an up-regulation of αIIb and fibrinogen isoforms in platelets from obese patients. A complementary platelet aggregation approach showed platelets from obese patients are hyper-reactive in response to collagen and collagen-related peptide (CRP), revealing the collagen receptor Glycoprotein VI (GPVI) signalling as one of the altered pathways. We also found the active form of Src (pTyr418) is up-regulated in platelets from obese individuals, which links proteomics to aggregation data. Moreover, we showed that CRP-activated platelets present higher levels of tyrosine phosphorylated PLCγ2 in obese patients, confirming alterations in GPVI signalling. In line with the above, flow cytometry studies show higher surface expression levels of total GPVI and GPVI-dimer in obese platelets, both correlating with BMI. Conclusions Our results suggest a higher activation state of SFKs-mediated signalling pathways in platelets from obese patients, with a primary involvement of GPVI signalling. Graphical abstract Image 1 Highlights • Higher expression of the active form of Src (pTyr418) in obese patients platelets. • Increased aggregation levels of obese platelets in response to GPVI activation. • GPVI-activated platelets show higher levels of tyr-phosphorylated PLCγ2 in obesity. • Higher surface expression levels of GPVI in obese platelets, correlating with BMI. [ABSTRACT FROM AUTHOR]

Expelling and hexane extraction are two typical processes for soybean oil production used in industry. The main issues for these two processes are the low efficiency and hazardous chemical problems respectively. Enzyme assisted aqueous extraction process (EAEP) was proposed to increase the efficiency without using organic solvent, which is replaced by water. The environmental impact analysis of these three processes are based on their mass flows, energy consumption and global warming potential. For mass flows, the environmental impact indices were calculated based on material flow of input and output components. Energy consumption was used to evaluate the carbon dioxide, other greenhouse gas (GHG) and criteria pollutants emissions by GREET models. According to our results, hexane extraction has the highest environmental impact due to the application of organic solvent. Expelling has the highest GHG and criteria pollutants emissions because of the high energy requirement for heat pressing processes. EAEP has similar environmental impacts to the expelling process, but it also lowers GHG and criteria pollutants emissions. EAEP has the potential to be a green process adopted by industry although a high energy intense pretreatment to produce finer soybean flakes for increasing oil recovery is still a challenge. [ABSTRACT FROM AUTHOR]

Isolated pea protein (IPP) dispersions (1%, w/v) were pretreated with high pressure (HP) of 200, 400, or 600 MPa for 5 min at 24 °C or high temperature (HT) for 30 min at 100 °C prior to hydrolysis with 1% (w/w) Alcalase. HP pretreatment of IPP at 400 and 600 MPa levels led to significantly ( P < 0.05) improved (>40%) oxygen radical absorption capacity (ORAC) of hydrolysates. 2,2-Diphenyl-1-picrylhydrazyl, superoxide radical and hydroxyl radical scavenging activities of pea protein hydrolysates were also significantly ( P < 0.05) improved (25%, 20%, and 40%, respectively) by HP pretreatment of IPP. Protein hydrolysates from HT IPP showed no ORAC, superoxide or hydroxyl scavenging activity but had significantly ( P < 0.05) improved (80%) ferric reducing antioxidant power. The protein hydrolysates had weaker antioxidant properties than glutathione but overall, the HP pretreatment was superior to HT pretreatment in facilitating enzymatic release of antioxidant peptides from IPP. [ABSTRACT FROM AUTHOR]

Background: Glycoprotein VI (GPVI) is the essential platelet collagen receptor in atherothrombosis, but its inhibition causes only a mild bleeding tendency. Thus, targeting this receptor has selective antithrombotic potential.Objectives: This study sought to compare compounds interfering with platelet GPVI-atherosclerotic plaque interaction to improve current antiatherothrombotic therapy.Methods: Human atherosclerotic plaque-induced platelet aggregation was measured in anticoagulated blood under static and arterial flow conditions (550/s, 1,100/s, and 1,500/s). Inhibition by dimeric GPVI fragment crystallizable region of IgG (Fc) masking GPVI binding sites on collagen was compared with that of 3 anti-GPVI antibodies: BLO8-1, a human domain antibody; 5C4, a fragment antigen-binding (Fab fragment) of monoclonal rat immunoglobulin G; and m-Fab-F, a human recombinant sFab against GPVI dimers.Results: GPVI-Fc reduced plaque-triggered platelet aggregation in static blood by 51%, BLO8-1 by 88%, and 5C4 by 93%. Under arterial flow conditions, BLO8-1 and 5C4 almost completely inhibited platelet aggregation while preserving platelet adhesion on plaque. Inhibition by GPVI-Fc, even at high concentrations, was less marked but increased with shear rate. Advanced optical imaging revealed rapid persistent GPVI-Fc binding to collagen under low and high shear flow, upstream and downstream of plaque fragments. At low shear particularly, platelets adhered in plaque flow niches to GPVI-Fc-free segments of collagen fibers and recruited other platelets onto aggregates via ADP and TxA2 release.Conclusions: Anti-GPVI antibodies inhibit atherosclerotic plaque-induced platelet aggregation under static and flow conditions more effectively than GPVI-Fc. However, potent platelet inhibition by GPVI-Fc at a higher shear rate (1,500/s) suggests localized antithrombotic efficacy at denuded or fissured stenotic high-risk lesions without systemic bleeding. The compound-specific differences have relevance for clinical trials targeting GPVI-collagen interaction combined with established antiplatelet therapies in patients with spontaneous plaque rupture or intervention-associated plaque injury. [ABSTRACT FROM AUTHOR]

Background Glycoprotein VI (GPVI) is the essential platelet collagen receptor in atherothrombosis, but its inhibition causes only a mild bleeding tendency. Thus, targeting this receptor has selective antithrombotic potential. Objectives This study sought to compare compounds interfering with platelet GPVI–atherosclerotic plaque interaction to improve current antiatherothrombotic therapy. Methods Human atherosclerotic plaque–induced platelet aggregation was measured in anticoagulated blood under static and arterial flow conditions (550/s, 1,100/s, and 1,500/s). Inhibition by dimeric GPVI fragment crystallizable region of IgG (Fc) masking GPVI binding sites on collagen was compared with that of 3 anti-GPVI antibodies: BLO8-1, a human domain antibody; 5C4, a fragment antigen-binding (Fab fragment) of monoclonal rat immunoglobulin G; and m-Fab-F, a human recombinant sFab against GPVI dimers. Results GPVI-Fc reduced plaque-triggered platelet aggregation in static blood by 51%, BLO8-1 by 88%, and 5C4 by 93%. Under arterial flow conditions, BLO8-1 and 5C4 almost completely inhibited platelet aggregation while preserving platelet adhesion on plaque. Inhibition by GPVI-Fc, even at high concentrations, was less marked but increased with shear rate. Advanced optical imaging revealed rapid persistent GPVI-Fc binding to collagen under low and high shear flow, upstream and downstream of plaque fragments. At low shear particularly, platelets adhered in plaque flow niches to GPVI-Fc–free segments of collagen fibers and recruited other platelets onto aggregates via ADP and TxA2 release. Conclusions Anti-GPVI antibodies inhibit atherosclerotic plaque-induced platelet aggregation under static and flow conditions more effectively than GPVI-Fc. However, potent platelet inhibition by GPVI-Fc at a higher shear rate (1,500/s) suggests localized antithrombotic efficacy at denuded or fissured stenotic high-risk lesions without systemic bleeding. The compound-specific differences have relevance for clinical trials targeting GPVI-collagen interaction combined with established antiplatelet therapies in patients with spontaneous plaque rupture or intervention-associated plaque injury. [ABSTRACT FROM AUTHOR]

Abstract: The objective of this study was to investigate natural antimicrobials including cranberry powder, dried vinegar and lemon juice/vinegar concentrate, and post-lethality interventions (lauric arginate, octanoic acid, thermal treatment and high hydrostatic pressure) for the control of Listeria monocytogenes on alternatively-cured frankfurters. Lauric arginate, octanoic acid, and high hydrostatic pressure (400MPa) reduced L. monocytogenes populations by 2.28, 2.03, and 1.88log10CFU per g compared to the control. L. monocytogenes grew in all post-lethality intervention treatments, except after a 600MPa high hydrostatic pressure treatment for 4 min. Cranberry powder did not inhibit the growth of L. monocytogenes, while a dried vinegar and a vinegar/lemon juice concentrate did. This study demonstrated the bactericidal properties of high hydrostatic pressure, octanoic acid and lauric arginate, and the bacteriostatic potential of natural antimicrobial ingredients such as dried vinegar and vinegar/lemon juice concentrate against L. monocytogenes. [Copyright &y& Elsevier]

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]

Abstract: High pressure processing of ovotransferrin was carried out to study the structural and physiochemical changes of ovotransferrin under various pressure levels. At pH 8 and pressures higher than 200MPa, a decrease in total sulfhydryl groups and an increase in surface hydrophobicity were observed along with a partial aggregation. A gradual shift of denaturation peak towards higher temperature was noticed up to 500MPa, leading to a total loss of the enthalpy of denaturation at pressures of 600 and 700MPa, where a significant decrease in intrinsic fluorescence was also observed. At pH 3, the ovotransferrin adopted a molten globule state, associated with a significant increase in surface hydrophobicity and reactive sulfhydryl content; structurally, no clear denaturation peaks in differential scanning calorimetry (DSC) were detected at any level of pressure treatment whereas a noticeable decrease in intrinsic fluorescence was evidenced up to 600MPa and then increased at 700MPa pressure treatment. Fourier transform infrared spectroscopy (FT-IR) revealed that the conformational structure were changed from helices, sheets, turns, and aggregated strand to mostly intermolecular β-sheets or aggregated strands at pH 8 at 200MPa but switched back to original structure at higher pressures. [Copyright &y& Elsevier]

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]

Abstract: Soybean insoluble fractions recovered from aqueous extraction processing (AEP) and enzyme-assisted AEP (EAEP) of full-fat soybean flakes (FFSF) and extruded FFSF were evaluated as a feedstock for the production of fermentable sugars using enzymes. Among the four insoluble fractions (AEP FFSF, EAEP FFSF, AEP extruded FFSF and EAEP extruded FFSF), the composition analysis revealed that the one recovered from EAEP of extruded FFSF had the highest glucan content, 16% [dry basis (db)], as compared to about 10% (db) for the other fractions. Thirty-three percent of the initial glucan of the insoluble recovered from AEP and EAEP of FFSF were converted into glucose using 33 FPU of Accellerase 1000/g-glucan. This saccharification yield was increased to 44% with extruded fibers. The higher saccharification yield of 49% was obtained at 45°C, 1% glucan loading, and 101 FPU/g-glucan enzymes loading after 27h of hydrolysis. [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]

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]

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]