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This study focuses on the enzymatic hydrolysis of hemoglobin, the main component of cruor that gives blood its red color in mammals. The antibacterial and antioxidant potentials of human hemoglobin hydrolysates were evaluated in comparison to bovine hemoglobin. The results showed strong antimicrobial activity of the peptide hydrolysates against six bacterial strains, independent of the initial substrate concentration level. The hydrolysates also showed strong antioxidant activity, as measured by four different tests. In addition, the antimicrobial and antioxidant activities of the human and bovine hemoglobin hydrolysates showed little or no significant difference, with only the concentration level being the determining factor in their activity. The results of the mass spectrometry study showed the presence of a number of bioactive peptides, the majority of which have characteristics similar to those mentioned in the literature. New bioactive peptides were also identified in human hemoglobin, such as the antibacterial peptides PTTKTYFPHF (α37-46), FPTTKTYFPH (α36-45), TSKYR (α137-141), and STVLTSKYR (α133-141), as well as the antioxidant TSKYR (α137-141). According to these findings, human hemoglobin represents a promising source of bioactive peptides beneficial to the food or pharmaceutical industries. [ABSTRACT FROM AUTHOR]

Cruor, the main component responsible for the red color of mammalian blood, contains 90% haemoglobin, a protein considered to be a rich source of bioactive peptides. The aim of the present study is to assess the potential of human hemoglobin as a source of bioactive peptides, compared with bovine hemoglobin, which has been extensively studied in recent years. More specifically, the study focused on the α137–141 fragment of bovine haemoglobin (TSKYR), a small (653 Da) hydrophilic antimicrobial peptide. In this work, the potential of human hemoglobin to contain bioactive peptides was first investigated in silico in comparison with bovine hemoglobin-derived peptides using bioinformatics tools. The blast results showed a high identity, 88% and 85% respectively, indicating a high similarity between the α and β chains. Peptide Cutter software was used to predict cleavage sites during peptide hydrolysis, revealing major conservation in the number and location of cleavage sites between the two species, while highlighting some differences. Some peptides were conserved, notably our target peptide (TSKYR), while others were specific to each species. Secondly, the two types of hemoglobin were subjected to similar enzymatic hydrolysis conditions (23 °C, pH 3.5), which showed that the hydrolysis of human hemoglobin followed the same reaction mechanism as the hydrolysis of bovine hemoglobin, the 'zipper' mechanism. Concerning the peptide of interest, α137–141, the RP-UPLC analyses showed that its identification was not affected by the increase in the initial substrate concentration. Its production was rapid, with more than 60% of the total α137–141 peptide production achieved in just 30 min of hydrolysis, reaching peak production at 3 h. Furthermore, increasing the substrate concentration from 1% to 10% (w/v) resulted in a proportional increase in α137–141 production, with a maximum concentration reaching 687.98 ± 75.77 mg·L−1, approximately ten-fold higher than that obtained with a 1% (w/v) concentration. Finally, the results of the UPLC-MS/MS analysis revealed the identification of 217 unique peptides in bovine hemoglobin hydrolysate and 189 unique peptides in human hemoglobin hydrolysate. Of these, 57 peptides were strictly common to both species. This revealed the presence of several bioactive peptides in both cattle and humans. Although some had been known previously, new bioactive peptides were discovered in human hemoglobin, such as four antibacterial peptides (α37–46 PTTKTYFPHF, α36–45 FPTTKTYFPH, α137–141 TSKYR, and α133–141 STVLTSKYR), three opioid peptides (α137–141 TSKYR,β31–40 LVVYPWTQRF,β32–40, VVYPWTQRF), an ACE inhibitor (β129–135 KVVAGVA), an anticancer agent (β33–39 VVYPWTQ), and an antioxidant (α137–141 TSKYR). To the best of our knowledge, these peptides have never been found in human hemoglobin before. [ABSTRACT FROM AUTHOR]

Bioinformatics software, allowing the identification of peptides by the comparison of peptide fragmentation spectra obtained by mass spectrometry versus targeted databases or directly by de novo sequencing, is now mandatory in peptidomics/proteomics approaches. Programming the identification software requires specifying, among other things, the mass measurement accuracy of the instrument and the digestion enzyme used with the number of missed cleavages allowed. Moreover, these software algorithms are able to identify a large number of post-translational modifications (PTMs). However, peptide and PTM identifications are challenging in the agrofood field due to non-specific cleavage sites of physiological- or food-grade enzymes and the number and location of PTMs. In this study, we show the importance of customized software programming to obtain a better peptide and PTM identification rate in the agrofood field. A gelatine product and one industrial gelatine hydrolysate from three different sources (beef, pork, and fish), each digested by simulated gastrointestinal digestion, MS-grade trypsin, or both, were used to perform the comparisons. Two main points are illustrated: (i) the impact of the set-up of specific enzyme versus no specific enzyme use and (ii) the impact of a maximum of six PTMs allowed per peptide versus the standard of three. Prior knowledge of the composition of the raw proteins is an important asset for better identification of peptide sequences. [ABSTRACT FROM AUTHOR]

Lacticaseicin 30 is one of the five bacteriocins produced by the Gram-positive Lacticaseibacillus paracasei CNCM I-5369. This 111 amino acid bacteriocin is noteworthy for being active against Gram-negative bacilli including Escherichia coli strains resistant to colistin. Prediction of the lacticaseicin 30 structure using the Alphafold2 pipeline revealed a largely helical structure including five helix segments, which was confirmed by circular dichroism. To identify the structural requirements of the lacticaseicin 30 activity directed against Gram-negative bacilli, a series of variants, either shortened or containing point mutations, was heterologously produced in Escherichia coli and assayed for their antibacterial activity against a panel of target strains including Gram-negative bacteria and the Gram-positive Listeria innocua. Lacticaseicin 30 variants comprising either the N-terminal region (amino acids 1 to 39) or the central and C-terminal regions (amino acids 40 to 111) were prepared. Furthermore, mutations were introduced by site-directed mutagenesis to obtain ten bacteriocin variants E6G, T7P, E32G, T33P, T52P, D57G, A74P, Y78S, Y93S and A97P. Compared to lacticaseicin 30, the anti-Gram-negative activity of the N-terminal peptide and variants E32G, T33P and D57G remained almost unchanged, while that of the C-terminal peptide and variants E6G, T7P, T52P, A74P, Y78S, Y93S and A97P was significantly altered. Finally, the N-terminal region was further shortened to keep only the first 20 amino acid part that was predicted to include the first helix. The anti-Gram-negative activity of this truncated peptide was completely abolished. Overall, this study shows that activity of lacticaseicin 30, one of the rare Gram-positive bacteriocins inhibiting Gram-negative bacteria, requires at least two helices in the N-terminal region and that the C-terminal region carries amino acids playing a role in modulation of the activity. Taken together, these data will help to design forthcoming variants of lacticaseicin 30 as promising therapeutic agents to treat infections caused by Gram-negative bacilli. [ABSTRACT FROM AUTHOR]

Dipeptidyl-peptidase IV (DPP-IV) plays an essential role in glucose metabolism by inactivating incretins. In this context, food-protein-derived DPP-IV inhibitors are promising glycemic regulators which may act by preventing the onset of type 2 diabetes in personalized nutrition. In this study, the DPP-IV-inhibitory potential of seven proteins from diverse origins was compared for the first time in vitro and in vivo in rat plasma after the intestinal barrier (IB) passage of the indigested proteins. The DPP-IV-inhibitory potentials of bovine hemoglobin, caseins, chicken ovalbumin, fish gelatin, and pea proteins were determined in rat plasma thirty minutes after oral administration. In parallel, these proteins, together with bovine whey and gluten proteins, were digested using the harmonized INFOGEST protocol adapted for proteins. The DPP-IV half-maximal inhibitory concentration (IC50) was determined in situ using Caco-2 cells. The DPP-IV-inhibitory activity was also measured after IB passage using a Caco2/HT29-MTX mixed-cell model. The peptide profiles were analyzed using reversed-phase high-performance liquid chromatography tandem mass spectrometry (RP-HPLC-MS/MS) with MS data bioinformatics management, and the IC50 of the identified peptides was predicted in silico. The in vitro and in vivo DPP-IV-inhibitory activity of the proteins differed according to their origin. Vegetable proteins and hemoglobin yielded the highest DPP-IV-inhibitory activity in vivo. However, no correlation was found between the in vivo and in vitro results. This may be partially explained by the differences between the peptidome analysis and the in silico predictions, as well as the study complexity. [ABSTRACT FROM AUTHOR]

This study investigated the antiradical and antioxidant potential of the three families of lipopeptides (i.e., surfactin, mycosubtilin, and plipastatin/fengycin) produced by Bacillus subtilis strains. The antiradical/antioxidant activities of highly purified lipopeptides were studied in acellular models using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, superoxide anion ( O 2. - ), hydrogen peroxide, (H2O2) and hydroxyl radical (HO.). At a lipopeptide concentration of 500 mg.L−1, the maximum inhibition of DPPH reached 22.88% (obtained for plipastatin). Moreover, the scavenging effects of O 2. - , H2O2, and HO. at the highest concentration tested (250 mg.L−1) were found to be 6, 21, and 3% for surfactin, 19, 9, and 15% for mycosubtilin, 21, 18, and 59% for plipastatin, 21, 31, and 61% for the mixture of surfactin/plipastatin, and 13, 16, and 15% for the mixture of surfactin/mycosubtilin, respectively. These results showed that plipastatin was the best candidate due to its antioxidant activities. [ABSTRACT FROM AUTHOR]

This study aimed to examine the ability of ulvan, a water-soluble polysaccharide from the green seaweed Ulva fasciata , to provide protection and induce resistance in wheat against the hemibiotrophic fungus Zymoseptoria tritici. Matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) analysis indicated that ulvan is mainly composed of unsaturated monosaccharides (rhamnose, rhamnose-3-sulfate, and xylose) and numerous uronic acid residues. In the greenhouse, foliar application of ulvan at 10 mg.ml–1 2 days before fungal inoculation reduced disease severity and pycnidium density by 45 and 50%, respectively. Ulvan did not exhibit any direct antifungal activity toward Z. tritici , neither in vitro nor in planta. However, ulvan treatment significantly reduced substomatal colonization and pycnidium formation within the mesophyll of treated leaves. Molecular assays revealed that ulvan spraying elicits, but does not prime, the expression of genes involved in several wheat defense pathways, including pathogenesis-related proteins (β-1,3-endoglucanase and chitinase), reactive oxygen species metabolism (oxalate oxidase), and the octadecanoid pathway (lipoxygenase and allene oxide synthase), while no upregulation was recorded for gene markers of the phenylpropanoid pathway (phenylalanine ammonia-lyase and chalcone synthase). Interestingly, the quantification of 83 metabolites from major chemical families using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) in both non-infectious and infectious conditions showed no substantial changes in wheat metabolome upon ulvan treatment, suggesting a low metabolic cost associated with ulvan-induced resistance. Our findings provide evidence that ulvan confers protection and triggers defense mechanisms in wheat against Z. tritici without major modification of the plant physiology. [ABSTRACT FROM AUTHOR]

Abstract Background Brain capillary endothelial cells (BCECs) form the physiological basis of the blood-brain barrier (BBB). The barrier function is (at least in part) due to well-known proteins such as transporters, tight junctions and metabolic barrier proteins (e.g. monoamine oxidase, gamma glutamyltranspeptidase and P-glycoprotein). Our previous 2-dimensional gel proteome analysis had identified a large number of proteins and revealed the major role of dynamic cytoskeletal remodelling in the differentiation of bovine BCECs. The aim of the present study was to elaborate a reference proteome of Triton X-100-soluble species from bovine BCECs cultured in the well-established in vitro BBB model developed in our laboratory. Results A total of 215 protein spots (corresponding to 130 distinct proteins) were identified by 2-dimensional gel electrophoresis, whereas over 350 proteins were identified by a shotgun approach. We classified around 430 distinct proteins expressed by bovine BCECs. Our large-scale gene expression analysis enabled the correction of mistakes referenced into protein databases (e.g. bovine vinculin) and constitutes valuable evidence for predictions based on genome annotation. Conclusions Elaboration of a reference proteome constitutes the first step in creating a gene expression database dedicated to capillary endothelial cells displaying BBB characteristics. It improves of our knowledge of the BBB and the key proteins in cell structures, cytoskeleton organization, metabolism, detoxification and drug resistance. Moreover, our results emphasize the need for both appropriate experimental design and correct interpretation of proteome datasets.

Vibrio bacteria, and particularly members of the Harveyi clade, are the causative agents of vibriosis. This disease is responsible for mass mortality events and important economic losses on aquaculture farms. Improvements in surveillance and diagnosis are needed to successfully manage vibriosis outbreaks. 16S rRNA gene sequencing is generally considered to be the gold standard for bacterial identification but the cost and long processing time make it difficult to apply for routine identification. In contrast, MALDI-TOF MS offers rapid diagnosis and is commonly used in veterinary laboratories today. The major limiting factor for using this technique is the low environmental bacterial diversity in the commonly available databases. Here, we demonstrate that the sole use of the commercially available Bruker BioTyper database is not fully adequate for identifying Vibrio bacteria isolated from aquaculture farms. We therefore developed a new in-house database named Luvibase, composed of 23 reference MALDI-TOF mass spectra profiles obtained from Vibrio collection strains, mostly belonging to the Harveyi clade. The comparison of the accuracy of MALDI-TOF MS profiling and 16S rRNA gene sequencing revealed a lack of resolution for 16S rRNA gene sequencing. In contrast, MALDI-TOF MS profiling proved to be a more reliable tool for resolving species-level variations within the Harveyi clade. Finally, combining the Luvibase with the Bruker ver.9.0.0.0 database, led to successful identification of 47 Vibrio isolates obtained from moribund abalone, seabass and oysters. Thus, the use of Luvibase allow for increased confidence in identifying Vibrio species belonging to the Harveyi clade. [ABSTRACT FROM AUTHOR]

Lignin is the second most abundant polymer after cellulose in lignocellulosic biomass. Its aromatic composition and recalcitrant nature make its valorization a major challenge for obtaining low molecular weight aromatics compounds with high value‐added from the enzymatic depolymerization of industrial lignins. The oxidation reaction of lignin polymer using laccases alone remains inefficient. Therefore, researches are focused on the use of a laccase‐mediator system (LMS) to facilitate enzymatic depolymerization. Until today, the LMS system was studied using water‐soluble lignin only (commercial lignins, modified lignins, or lignin model compounds). This work reports a study of three LMS systems to depolymerize the three major industrial lignins (organosolv lignin, Kraft lignin, and sodium lignosulfonate). We show that an enzymatic depolymerization of these lignins can be achieved by LMS using laccase from Trametes versicolor, 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) diammonium salt as mediator and a cosolvent (25% of 1,4‐dioxane) to enhance the solubilization of lignins. [ABSTRACT FROM AUTHOR]

Copepods represent a major source of food for many aquatic species of commercial interest for aquaculture such as mysis shrimp and early stages of fishes. For the purpose of this study, the culturable mesophilic bacterial flora colonizing Acartia tonsa copepod eggs was isolated and identified. A total of 175 isolates were characterized based on their morphological and biochemical traits. The majority of these isolates (70%) were Gram-negative bacteria. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was used for rapid identification of bacterial isolates. Here, 58% of isolates were successfully identified at the genus level and among them, 54% were identified at the species level. These isolates belong to 12 different genera and 29 species. Five strains, identified as Bacillus pumilus, named 18 COPS, 35A COPS, 35R COPS, 38 COPS, and 40A COPS, showed strong antagonisms against several potential fish pathogens including Vibrio alginolyticus, V. anguillarum, Listeria monocytogenes, and Staphylococcus aureus. Furthermore, using a differential approach, we show that the antimicrobial activity of the 35R COPS strain is linked primarily to the production of antimicrobial compounds of the amicoumacin family, as demonstrated by the specific UV-absorbance and the MS/MS fragmentation patterns of these compounds. [ABSTRACT FROM AUTHOR]

The article discusses a study which investigated the effect of receptor for advanced glycation end-products (RAGE) ligands and anti-sRAGE autoantibodies on soluble form of the receptor for advanced glycation end-products (sRAGE) quantification. Topics explored include haemodialysis (HD) patients, biomarkers, and enzyme-linked immunosorbent assay (ELISA). Among the featured RAGE ligands include carboxymethyllysine, S100 proteins, and high mobility group protein B1 (HMGB1).

Although the physiological properties of the blood-brain barrier (BBB) are relatively well known, the phenotype of the component brain capillary endothelial cells (BCECs) has yet to be described in detail. Likewise, the molecular mechanisms that govern the establishment and maintenance of the BBB are largely unknown. Proteomics can be used to assess quantitative changes in protein levels and identify proteins involved in the molecular pathways responsible for cellular differentiation. Using the well-established in vitro BBB model developed in our laboratory, we performed a differential nano- LC MALDI-TOF/TOF-MS study of Triton X-100-soluble protein species from bovine BCECs displaying either limited BBB functions or BBB functions re-induced by glial cells. Due to the heterogeneity of the crude extract, we increased identification yields by applying a repeatable, reproducible fractionation process based on the proteins' relative hydrophobicity. We present proteomic and biochemical evidence to show that tissue non-specific alkaline phosphatase (TNAP) and Eps15 homology domain-containing protein 1(EDH1) are over-expressed by bovine BCECs after the re-induction of BBB properties. We discuss the impact of these findings on current knowledge of endothelial and BBB permeability. [ABSTRACT FROM AUTHOR]

Background: Brain capillary endothelial cells (BCECs) form the physiological basis of the blood-brain barrier (BBB). The barrier function is (at least in part) due to well-known proteins such as transporters, tight junctions and metabolic barrier proteins (e.g. monoamine oxidase, gamma glutamyltranspeptidase and P-glycoprotein). Our previous 2-dimensional gel proteome analysis had identified a large number of proteins and revealed the major role of dynamic cytoskeletal remodelling in the differentiation of bovine BCECs. The aim of the present study was to elaborate a reference proteome of Triton X-100-soluble species from bovine BCECs cultured in the wellestablished in vitro BBB model developed in our laboratory. Results: A total of 215 protein spots (corresponding to 130 distinct proteins) were identified by 2-dimensional gel electrophoresis, whereas over 350 proteins were identified by a shotgun approach. We classified around 430 distinct proteins expressed by bovine BCECs. Our large-scale gene expression analysis enabled the correction of mistakes referenced into protein databases (e.g. bovine vinculin) and constitutes valuable evidence for predictions based on genome annotation. Conclusions: Elaboration of a reference proteome constitutes the first step in creating a gene expression database dedicated to capillary endothelial cells displaying BBB characteristics. It improves of our knowledge of the BBB and the key proteins in cell structures, cytoskeleton organization, metabolism, detoxification and drug resistance. Moreover, our results emphasize the need for both appropriate experimental design and correct interpretation of proteome datasets. [ABSTRACT FROM AUTHOR]

Abstract: The blood–brain barrier (BBB) contributes to the brain homeostasis by regulating the passage of endogenous and exogenous compounds. This function is in part due to well-known proteins such as tight junction proteins, plasma membrane transporters and metabolic barrier proteins. Over the last decade, genomics and proteomics have emerged as supplementary tools for BBB research. The development of genomic and proteomic technologies has provided several means to extend the BBB knowledge and to investigate additional routes for the bypass of this barrier. These profiling technologies have been used on BBB models to decipher the physiological characteristics and, under stress conditions, to understand the molecular mechanisms of brain diseases. In this review, we will report and discuss the genomic and proteomic studies recently carried out to enhance the understanding of BBB features. [Copyright &y& Elsevier]