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Chondroitin sulfate (CS), a glycosaminoglycan, supports health through various physiological functions, including tissue protection, bone growth, and skin aging prevention. It also contributes to anticoagulant or anti-inflammatory processes, with its primary clinical use being osteoarthritis treatment. This study presents the results of the valorization of lipids and CS, both extracted from salmon co-products through enzymatic processes. The polar lipids, naturally rich in long-chain fatty acids (docosahexaenoic acid DHA C22:6 n-3 and eicosapentaenoic acid EPA C20:5 n-3), and the CS, primarily located in the nasal cartilage, were separated and concentrated before being characterized using various techniques to determine functional and lipid composition. These compounds were then used to formulate liposomes of 63 to 95 nm in size composed of 19.38% of DHA and 7.44% of EPA and encapsulating CS extract with a Δdi-4S/Δdi-6S ratio of 0.53 at 2 weight masses (10–30 kDa and >30 kDa) or CS standard all at two different concentrations. Liposomes were tested on human chondrocytes in inflamed conditions. Thus, compatibility tests, the expression of various inflammation markers at transcriptional and molecular levels, nitrites, and the amount of collagenase produced were analyzed. The results showed that CS, in synergy with the liposomes, played a positive role in combating chondrocyte inflammation even at a low concentration. [ABSTRACT FROM AUTHOR]

The current study aimed to investigate the chemical composition, antioxidant, antibacterial, and cytotoxic properties of three extracts (hexane, dichloromethane, and methanol) from Cynoglossum tubiflorus. The composition of the methanolic extract was elucidated using HPLC-HESI-MS/MS analysis. The antioxidant effect was examined using NO, DPPH, FRAP, and TAC assays. Antimicrobial activity was evaluated by broth microdilution using various bacterial strains such as S. aureus, S. epidermidis, P. aeruginosa, E. coli, and K. pneumoniae. Structural disruptions in Gram-positive bacteria were visualized using scanning electron microscopy (SEM). Cytotoxic effects were evaluated on human MRC-5 in culture according to the MTT assay. The outcomes suggest that methanol extract contained a high amount of phenolic compounds (254.35 ± 0.360 mg GAE/g DE and 211.59 ± 0.939 mg QE/g DE). By applying the HPLC-HESI-MS/MS analysis, 32 compounds were identified, including phenolic acids, flavonoids, lignans, and fatty acids. This extract showed strong antioxidant (IC50 = 0.043 ± 0.001 mg/mL) and antimicrobial (MIC = 156 µg/mL) activities. The SEM suggests that cells exhibited membrane distortions characterized by surface depressions and alterations in bacterial shape, including dents, when compared to untreated cells. The in vitro cytotoxicity effect on human MRC-5 cells showed no toxicity effects at a concentration of 600 µg/mL. In silico analysis predicted low toxicity for all tested compounds across four different administration routes. This research indicates that this plant could be explored as a powerful source of natural drugs to target pathogens, with applications in the food, pharmaceutical, and medical industries. [ABSTRACT FROM AUTHOR]

Various peptides can be obtained through protein enzymatic hydrolysis. Immobilized metal ion affinity chromatography (IMAC) is one of the methods which can be used to separate metal chelating peptides (MCPs) in a hydrolysate mixture. In this context, this work aims to understand deeply the interactions in IMAC and surface plasmon resonance (SPR) in order to validate experimentally the analogy between both technologies and to be further able to perform IMAC modeling in the next work using peptide sorption isotherm parameters obtained from SPR. Indeed, chromatography modeling can be used to predict separation of MCPs in IMAC and the knowledge of peptide sorption isotherm obtained from SPR is a crucial step. For this purpose, 22 peptides were selected and investigated in IMAC using HisTrap X-Ni2+ and HiFliQ NTA-Ni2+ columns and were also studied in SPR as well. Results showed that peptides with histidine residues had good affinity to Ni2+, while the high positive charge of peptides was responsible of ionic interactions. Further, most of the peptides with good retention time in IMAC showed a good affinity in SPR as well, which validated experimentally the SPR-IMAC analogy. [ABSTRACT FROM AUTHOR]

To mobilize nutrients entrapped into minerals and rocks, heterotrophic bacteria living in nutrient-poor environments have developed different mechanisms based mainly on acidolysis and chelation. However, the genetic bases of these mechanisms remain unidentified. To fill this gap, we considered the model strain Caballeronia mineralivorans PML1(12) known to be effective at weathering. Based on its transcriptomics and proteomics responses in Fe-depleted conditions, we pointed a cluster of genes differentially expressed and putatively involved in the production of siderophores. In this study, we report the characterization of this gene region coding for the production of a non-ribosomal peptide synthetase-independent siderophore (NIS). Targeted mutagenesis associated with functional assays and liquid chromatography coupled to high-resolution tandem mass spectrometry demonstrated the production of a single siderophore, identified as rhizobactin. This siderophore represents the first NIS containing malic acid in its structure. The evidence for the implication of rhizobactin in mineral weathering was demonstrated during a hematite dissolution assay. This study provides the first demonstration of the synthesis of a NIS in the genus Caballeronia and its involvement in mineral weathering. Our conclusions reinforce the idea that strain PML1(12) is particularly well adapted to nutrient-poor environments. [ABSTRACT FROM AUTHOR]

The aim of the present work was to obtain microbial lipids (single-cell oils and SCOs) from oleaginous yeast cultivated on biodiesel-derived glycerol and subsequently proceed to the enzymatic synthesis of high-value biosurfactant-type molecules in an aqueous medium, with SCOs implicated as acyl donors (ADs). Indeed, the initial screening of five non-conventional oleaginous yeasts revealed that the most important lipid producer was the microorganism Cryptococcus curvatus ATCC 20509. SCO production was optimised according to the nature of the nitrogen source and the initial concentration of glycerol (Glyc0) employed in the medium. Lipids up to 50% w/w in dry cell weight (DCW) (SCOmax = 6.1 g/L) occurred at Glyc0 ≈ 70 g/L (C/N ≈ 80 moles/moles). Thereafter, lipids were recovered and were subsequently used as ADs in the N-acylation reaction catalysed by aminoacylases produced from Streptomyces ambofaciens ATCC 23877 under aqueous conditions, while Candida antarctica lipase B (CALB) was used as a reference enzyme. Aminoacylases revealed excellent activity towards the synthesis of acyl-lysine only when free fatty acids (FAs) were used as the AD, and the rare regioselectivity in the α-amino group, which has a great impact on the preservation of the functional side chains of any amino acids or peptides. Aminoacylases presented higher α-oleoyl-lysine productivity and final titer (8.3 g/L) with hydrolysed SCO than with hydrolysed vegetable oil. The substrate specificity of both enzymes towards the three main FAs found in SCO was studied, and a new parameter was defined, viz., Specificity factor (Sf), which expresses the relative substrate specificity of an enzyme towards a FA present in a FA mixture. The Sf value of aminoacylases was the highest with palmitic acid in all cases tested, ranging from 2.0 to 3.0, while that of CALB was with linoleic acid (0.9–1.5). To the best of our knowledge, this is the first time that a microbial oil has been successfully used as AD for biosurfactant synthesis. This bio-refinery approach illustrates the concept of a state-of-the-art combination of enzyme and microbial technology to produce high-value biosurfactants through environmentally friendly and economically sound processes. [ABSTRACT FROM AUTHOR]

The ferulic acid (FA)-oxidation by Myceliophthora thermophila laccase was performed in phosphate buffer at 30 °C and pH 7.5 as an eco-friendly procedure. LC-MS analysis showed that oxidation products were four dehydrodimers (P1, P2, P3, P5) at MM = 386 g/mol, two dehydrotetramers (P6, P7) at MM = 770 g/mol and one decarboxylated dehydrodimer (P4) at MM = 340 g/mol. Structural characterization showed that FA-dehydrodimers were symmetric for P1 and P5 while asymmetric for P2, P3 and P4. Physicochemical characterization showed that oxidation products presented a higher lipophilicity than that of FA. Moreover, symmetric dimers and tetra dimers had a higher melting point compared to FA and its asymmetric dimers. Antioxidant and anti-proliferative assessments indicated that enzymatic oligomerization increased antioxidant and anti-proliferative properties of oxidation products for P2, P3 and P6 compared to FA. Finally, this enzymatic process in water could produce new molecules, having good antiradical and anti-proliferative activities. [ABSTRACT FROM AUTHOR]

Summary: Mineral weathering by microorganisms is considered to occur through a succession of mechanisms based on acidification and chelation. While the role of acidification is established, the role of siderophores is difficult to disentangle from the effect of the acidification. We took advantage of the ability of strain Collimonas pratensis PMB3(1) to weather minerals but not to acidify depending on the carbon source to address the role of siderophores in mineral weathering. We identified a single non‐ribosomal peptide synthetase (NRPS) responsible for siderophore biosynthesis in the PMB3(1) genome. By combining iron‐chelating assays, targeted mutagenesis and chemical analyses (HPLC and LC‐ESI‐HRMS), we identified the siderophore produced as malleobactin X and how its production depends on the concentration of available iron. Comparison with the genome sequences of other collimonads evidenced that malleobactin production seems to be a relatively conserved functional trait, though some collimonads harboured other siderophore synthesis systems. We also revealed by comparing the wild‐type strain and its mutant impaired in the production of malleobactin that the ability to produce this siderophore is essential to allow the dissolution of hematite under non‐acidifying conditions. This study represents the first characterization of the siderophore produced by collimonads and its role in mineral weathering. [ABSTRACT FROM AUTHOR]

Pili are polymeric proteins located at the cell surface of bacteria. These filamentous proteins play a pivotal role in bacterial adhesion with the surrounding environment. They are found both in Gram-negative and Gram-positive bacteria but differ in their structural organization. Purifying these high molecular weight proteins is challenging and has certainly slowed down their characterization. Here, we propose a chromatography-based protocol, mainly relying on multimodal chromatography (core bead technology using Capto Core 700 resin), to purify sortase-dependent SpaCBA pili from the probiotic strain Lacticaseibacillus rhamnosus GG (LGG). Contrary to previously published methods, this purification protocol does not require specific antibodies nor complex laboratory equipment, including for the multimodal chromatography step, and provides high degree of protein purity. No other proteins were detectable by SDS-PAGE and the 260/280 nm ratio (∼0.6) of the UV spectrum confirmed the absence of any other co-purified macromolecules. One can obtain ∼50 μg of purified pili, starting from 1 L culture at OD600nm ≈ 1, in 2–3 working days. This simple protocol could be useful to numerous laboratories to purify pili from LGG easily. Therefore, the present work should boost specific studies dedicated to LGG SpaCBA pili and the characterization of the interactions occurring with their protein partners at the molecular level. Moreover, this straightforward purification process might be extended to the purification of sortase-dependant pili from other Gram-positive bacteria. [ABSTRACT FROM AUTHOR]

Some metal‐chelating peptides have antioxidant properties, with potential nutrition, health, and cosmetics applications. This study aimed to simulate their separation on immobilized metal ion affinity chromatography from their affinity constant for immobilized metal ion determined in surface plasmon resonance, both technics are based on peptide‐metal ion interactions. In our approach, first, the affinity constant of synthetic peptides was determined by surface plasmon resonance and used as input data to numerically simulate the chromatographic separation with a transport‐dispersive model based on Langmuir adsorption isotherm. Then, chromatographic separation was applied on the same peptides to determine their retention time and compare this experimental tR with the simulated tR obtained from simulation from surface plasmon resonance data. For the investigated peptides, the relative values of tR were comparable. Hence, our study demonstrated the pertinence of such numerical simulation correlating immobilized metal ion affinity chromatography and surface plasmon resonance. [ABSTRACT FROM AUTHOR]

The cell‐specific growth rate (µ) is a critical process parameter for antibody production processes performed by animal cell cultures, as it describes the cell growth and reflects the cell physiological state. When there are changes in these parameters, which are indicated by variations of µ, the synthesis and the quality of antibodies are often affected. Therefore, it is essential to monitor and control the variations of µto assure the antibody production and achieve high product quality. In this study, a novel approach for on‐line estimation of µ was developed based on the process analytical technology initiative by using an in situ dielectric spectroscopy. Critical moments, such as significant µ decreases, were successfully detected by this method, in association with changes in cell physiology as well as with an accumulation of nonglycosylated antibodies. Thus, this method was used to perform medium renewals at the appropriate time points, maintaining the values of µ close to its maximum. Using this method, we demonstrated that the physiological state of cells remained stable, the quantity and the glycosylation quality of antibodies were assured at the same time, leading to better process performances compared with the reference feed‐harvest cell cultures carried out by using off‐line nutrient measurements. The cell‐specific growth rate (μ) is a critical process parameter for antibody production by animal cell cultures, as it reflects the cell physiological state of which changes often affect synthesis and quality of antibodies. A novel approach for on‐line estimation of μ was developed by using in situ dielectric spectroscopy and used to control medium renewals during feed‐harvest cultures. This on‐line strategy allowed to maintain an appropriate cell physiological state and consequently to assure the antibody production and glycosylation quality. [ABSTRACT FROM AUTHOR]

Abstract: The presence of aminoacylase activities was investigated in a crude extract of Streptomyces ambofaciens ATCC23877. First activities catalyzing the hydrolysis of N‐α or ε‐acetyl‐L‐lysine were identified. Furthermore, the acylation of lysine and different peptides was studied and compared with results obtained with lipase B of Candida antarctica (CALB). Different regioselectivities were demonstrated for the two classes of enzymes. CALB was able to catalyze acylation only on the ε‐position whereas the crude extract from S. ambofaciens possessed the rare ability to catalyze the N‐acylation on the α‐position of the lysine or of the amino‐acid in N‐terminal position of peptides. Two genes, SAM23877_1485 and SAM23877_1734, were identified in the genome of Streptomyces ambofaciens ATCC23877 whose products show similarities with the previously identified aminoacylases from Streptomyces mobaraensis. The proteins encoded by these two genes were responsible for the major aminoacylase hydrolytic activities. Furthermore, we show that the hydrolysis of N‐α‐acetyl‐L‐lysine could be attributed to the product of SAM23877_1734 gene. [ABSTRACT FROM AUTHOR]

The glycosylation of therapeutic monoclonal antibodies (mAbs), a known critical quality attribute, is often greatly modified during the production process by animal cells. It is essential for biopharmaceutical industries to monitor and control this glycosylation. However, current glycosylation characterization techniques involve time‐ and labor‐intensive analyses, often carried out at the end of the culture when the product is already synthesized. This study proposes a novel methodology for real‐time monitoring of antibody glycosylation site occupancy using Raman spectroscopy. It was first observed in CHO cell batch culture that when low nutrient concentrations were reached, a decrease in mAb glycosylation was induced, which made it essential to rapidly detect this loss of product quality. By combining in situ Raman spectroscopy with chemometric tools, efficient prediction models were then developed for both glycosylated and nonglycosylated mAbs. By comparing variable importance in projection profiles of the prediction models, it was confirmed that Raman spectroscopy is a powerful method to distinguish extremely similar molecules, despite the high complexity of the culture medium. Finally, the Raman prediction models were used to monitor batch and feed‐harvest cultures in situ. For the first time, it was demonstrated that the concentrations of glycosylated and nonglycosylated mAbs could be successfully and simultaneously estimated in real time with high accuracy, including their sudden variations due to medium exchanges. Raman spectroscopy can thus be considered as a promising PAT tool for feedback process control dedicated to on‐line optimization of mAb quality. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:486–493, 2018 [ABSTRACT FROM AUTHOR]

The objective of this paper is on the one hand to study the effect of heat processing (30 to 130°C for 2 h) on the stability and antioxidant activity of six flavonoids (rutin, naringin, eriodictyol, mesquitol, luteolin, and luteolin 7-O glucoside), and on the other hand to establish the relation structure-activity-stability of these compounds. The dependency on temperature of the six kinetics was well described by the Arrhenius law and the main parameters provided by this model are determined and compared in this paper. Activation energies found were 107.3 kJ/mol for rutin, 100.6 for naringin, 33.3 for mesquitol, 68.2 for eriodictyol, 51.4 for luteolin, and 120 for luteolin 7-O-glucoside. The data collected showed that glycosylated flavonoids are more resistant than aglycon flavonoids to heat treatment. Moreover, it was also observed that despite the total degradation of some flavonoids, the treated solutions still have an antioxidant activity. Practical application Flavonoids are mainly consumed in processed foods. Thus, flavonoids often undergo heat processing. Knowing the thermal stability and the evolution of their antioxidant activity are particularly relevant in the field of food processing. The results of this study allow information to be collected on the relationship between heat stability-flavonoid structure and antioxidant activity. This information will be useful for the formulation of new food products or for the design of new food processes. [ABSTRACT FROM AUTHOR]

Background: Polyunsaturated fatty acids play a crucial role in neuronal function, and the modification of these compounds in the brain could have an impact on neurodegenerative diseases such as Alzheimer's disease. Despite the fact that arachidonic acid is the second foremost polyunsaturated fatty acid besides docosahexaenoic acid, its role and the regulation of its transfer and mobilization in the brain are poorly known. Methods: Two groups of 39 adult male BALB/c mice were fed with an arachidonic acid-enriched diet or an oleic acid-enriched diet, respectively, for 12 weeks. After 10 weeks on the diet, mice received intracerebroventricular injections of either NaCl solution or amyloid-β peptide (Aβ) oligomers. Y-maze and Morris water maze tests were used to evaluate short- and long-term memory. At 12 weeks on the diet, mice were killed, and blood, liver, and brain samples were collected for lipid and protein analyses. Results: We found that the administration of an arachidonic acid-enriched diet for 12 weeks induced short-term memory impairment and increased deleterious effects of Aβ oligomers on learning abilities. These cognitive alterations were associated with modifications of expression of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, postsynaptic density protein 95, and glial fibrillary acidic protein in mouse cortex or hippocampus by the arachidonic acid-enriched diet and Aβ oligomer administration. This diet also led to an imbalance between the main ω-6 fatty acids and the ω-3 fatty acids in favor of the first one in erythrocytes and the liver as well as in the hippocampal and cortical brain structures. In the cortex, the dietary arachidonic acid also induced an increase of arachidonic acid-containing phospholipid species in phosphatidylserine class, whereas intracerebroventricular injections modified several arachidonic acid- and docosahexaenoic acid-containing species in the four phospholipid classes. Finally, we observed that dietary arachidonic acid decreased the expression of the neuronal form of acyl-coenzyme A synthetase 4 in the hippocampus and increased the cytosolic phospholipase A2 activation level in the cortices of the mice. Conclusions: Dietary arachidonic acid could amplify Aβ oligomer neurotoxicity. Its consumption could constitute a risk factor for Alzheimer's disease in humans and should be taken into account in future preventive strategies. Its deleterious effect on cognitive capacity could be linked to the balance between arachidonic acid-mobilizing enzymes. [ABSTRACT FROM AUTHOR]

ContextCatharanthus roseus(L.) G. Don (Apocynaceae) is still one of the most important sources ofterpene indole alkaloids including anticancer and hypertensive drugs as vincristine and vinblastine. These final compounds have complex pathway and many enzymes are involved in their biosynthesis. Indeed, ajmalicine and catharanthine are important precursors their increase can lead to enhance levels of molecules of interest. ObjectiveThis study aims at selecting the highest yield of hairy root line(s) and at identifying best times for further treatments. We study kinetics growth and alkaloids (ajmalicine and catharanthine) accumulation of three selected hairy root lines during the culture cycle in order to determine the relationship between biomass production and alkaloids accumulation. Materials and methodsComparative analysis has been carried out on three selected lines ofCatharanthus roseushairy roots (LP10, LP21 and L54) for their kinetics of growth and the accumulation of ajamalicine and catharanthine, throughout a 35-day culture cycle. The methanolic extract for each line in different times during culture cycle is analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). ResultsMaximum accumulation of the alkaloids is recorded for LP10 line in which the peak of ajmalicine and catharanthine accumulation reached to 3.8 and 4.3 mg/g dry weight (DW), respectively. This increase coincides with an exponential growth phase. Discussion and conclusionOur results suggest that the evolution of accumulation of ajmalicine and catharanthine are positively correlated with the development of the biomass growth. Significantly, for LP10 line the most promising line to continue optimizing the production of TIAs. Additionally, the end of exponential phase remains the best period for elicitor stimuli. [ABSTRACT FROM PUBLISHER]

We describe the impact of two propeptides and PedC on the production yield and the potency of recombinant pediocins produced in L actococcus lactis. On the one hand, the sequences encoding the propeptides SD or LEISSTCDA were inserted between the sequence encoding the signal peptide of Usp45 and the structural gene of the mature pediocin PA-1. On the other hand, the putative thiol-disulfide oxidoreductase PedC was coexpressed with pediocin. The concentration of recombinant pediocins produced in supernatants was determined by enzyme-linked immunosorbent assay. The potency of recombinant pediocins was investigated by measuring the minimal inhibitory concentration by agar well diffusion assay. The results show that propeptides SD or LEISSTCDA lead to an improved secretion of recombinant pediocins with apparently no effect on the antibacterial potency and that PedC increases the potency of recombinant pediocin. To our knowledge, this study reveals for the first time that pediocin tolerates fusions at the N-terminal end. Furthermore, it reveals that only expressing the pediocin structural gene in a heterologous host is not sufficient to get an optimal potency and requires the accessory protein PedC. In addition, it can be speculated that PedC catalyses the correct formation of disulfide bonds in pediocin. [ABSTRACT FROM AUTHOR]

The phosphopantetheinyl transferases (PPTases) are responsible for the activation of the carrier protein domains of the polyketide synthases (PKS), non ribosomal peptide synthases (NRPS) and fatty acid synthases (FAS). The analysis of the Streptomyces ambofaciens ATCC23877 genome has revealed the presence of four putative PPTase encoding genes. One of these genes appears to be essential and is likely involved in fatty acid biosynthesis. Two other PPTase genes, samT0172 (alpN) and samL0372, are located within a type II PKS gene cluster responsible for the kinamycin production and an hybrid NRPS-PKS cluster involved in antimycin production, respectively, and their products were shown to be specifically involved in the biosynthesis of these secondary metabolites. Surprisingly, the fourth PPTase gene, which is not located within a secondary metabolite gene cluster, appears to play a pleiotropic role. Its product is likely involved in the activation of the acyl- and peptidyl-carrier protein domains within all the other PKS and NRPS complexes encoded by S. ambofaciens. Indeed, the deletion of this gene affects the production of the spiramycin and stambomycin macrolide antibiotics and of the grey spore pigment, all three being PKS-derived metabolites, as well as the production of the nonribosomally produced compounds, the hydroxamate siderophore coelichelin and the pyrrolamide antibiotic congocidine. In addition, this PPTase seems to act in concert with the product of samL0372 to activate the ACP and/or PCP domains of the antimycin biosynthesis cluster which is also responsible for the production of volatile lactones. [ABSTRACT FROM AUTHOR]

Prior studies on membranes have reported that unsaturations affect important bilayers properties, but most of these studies have been based on single-component membranes. This report compares the properties of a phosphatidylcholine (PC) membrane enriched in long chain PUFA (LC-PUFA), extracted and purified from salmon heads ( Salmo salar), with those of other multicomponent PC membranes from egg and soybean. Detailed description of the composition, the structural parameters, the thermal behavior, the hydration, and monolayer properties are given. Hydration properties of salmon PC showed greater adsorbance of water compared to egg and soybean PC. A greater adsorption of water was also observed for salmon PC multibilayers in bulk water before demixing compared to egg and soybean PC. An interesting feature concerns the evolution of area per lipid when increasing the water content where salmon PC showed a relative constant molecular area upon hydration. Another specific feature was the ability of salmon PC monolayer to occupy smaller molecular area upon compression than do egg and soybean PC. We hypothesize that specific hydration properties at the interface and enhanced packing properties of the LC-PUFA are responsible for this particular behavior of salmon PC membrane. [ABSTRACT FROM AUTHOR]

In the original version of this Article, the final concentration of riboflavin in the supplemented LB medium for recombinant LkcE expression was incorrectly stated as 1 g L−1 (this was the concentration of the stock solution) and should have read 10-50 mg L−1. This error has been corrected in both the PDF and HTML versions of the Article. [ABSTRACT FROM AUTHOR]