Relationship between hemolysis and lipid oxidation in red blood cell-spiked fish muscle; dependance on pH and blood plasma.

The relationship between hemolysis and lipid oxidation was explored in red blood cell (RBCs)-spiked washed cod mince (WCM). At pH 6.8 and 3 ± 1 °C, intact RBCs (71 µM Hb) delayed lipid oxidation by 1 day compared to WCM with partly or fully lysed RBCs which oxidized immediately. Intact RBCs also lowered peak peroxide value (PV) and thiobarbituric acid reactive substances (TBARS) with up to 59.5% and 48.1%, respectively. Adding 3% (v/w) blood plasma to RBC-spiked WCM delayed the lipid oxidation onset from 1 to 3-4 days without delaying hemolysis. At pH 6.4 the oxidation onset in RBC-WCM was the same as for pH 6.8 while at pH 7.2-7.6 lipid oxidation was suppressed for 7 days. Micrographs revealed RBC-lysis from day 2 at pH 6.4 but at pH 7.6, RBC stayed intact for ≥ 7 days. Thus, assuring presence of plasma-derived antioxidants and/or elevating muscle pH to avoid hemolysis can aid valorization of blood rich underutilized fish raw materials.

Personalized tissue-engineered veins - long term safety, functionality and cellular transcriptome analysis in large animals.

Tissue engineering is a promising methodology to produce advanced therapy medicinal products (ATMPs). We have developed personalized tissue engineered veins (P-TEV) as an alternative to autologous or synthetic vascular grafts utilized in reconstructive vein surgery. Our hypothesis is that individualization through reconditioning of a decellularized allogenic graft with autologous blood will prime the tissue for efficient recellularization, protect the graft from thrombosis, and decrease the risk of rejection. In this study, P-TEVs were transplanted to vena cava in pig, and the analysis of three veins after six months, six veins after 12 months and one vein after 14 months showed that all P-TEVs were fully patent, and the tissue was well recellularized and revascularized. To confirm that the ATMP product had the expected characteristics one year after transplantation, gene expression profiling of cells from P-TEV and native vena cava were analyzed and compared by qPCR and sequencing. The qPCR and bioinformatics analysis confirmed that the cells from the P-TEV were highly similar to the native cells, and we therefore conclude that P-TEV is functional and safe in large animals and have high potential for use as a clinical transplant graft.

Personalized tissue-engineered arteries as vascular graft transplants: A safety study in sheep.

Patients with cardiovascular disease often need replacement or bypass of a diseased blood vessel. With disadvantages of both autologous blood vessels and synthetic grafts, tissue engineering is emerging as a promising alternative of advanced therapy medicinal products for individualized blood vessels. By reconditioning of a decellularized blood vessel with the recipient's own peripheral blood, we have been able to prevent rejection without using immunosuppressants and prime grafts for efficient recellularization in vivo. Recently, decellularized veins reconditioned with autologous peripheral blood were shown to be safe and functional in a porcine in vivo study as a potential alternative for vein grafting. In this study, personalized tissue engineered arteries (P-TEA) were developed using the same methodology and evaluated for safety in a sheep in vivo model of carotid artery transplantation. Five personalized arteries were transplanted to carotid arteries and analyzed for safety and patency as well as with histology after four months in vivo. All grafts were fully patent without any occlusion or stenosis. The tissue was well cellularized with a continuous endothelial cell layer covering the luminal surface, revascularized adventitia with capillaries and no sign of rejection or infection. In summary, the results indicate that P-TEA is safe to use and has potential as clinical grafts.

Bioprocessed Brewers' Spent Grain Improves Nutritional and Antioxidant Properties of Pasta.

Brewers' spent grain (BSG), the by-product of brewing, was subjected to a xylanase treatment followed by fermentation with Lactiplantibacillus plantarum PU1. Bioprocessed BSG has been used as ingredient to obtain a fortified semolina pasta which can be labeled as "high fiber" and "source of protein" according to the European Community Regulation No. 1924/2006. Compared to native BSG, the use of bioprocessed BSG led to higher protein digestibility and quality indices (essential amino acid index, biological value, protein efficiency ratio, nutritional index), as well as lower predicted glycemic index. Bioprocessing also improved the technological properties of fortified pasta. Indeed, brightfield and confocal laser scanning microscopy revealed the formation of a more homogeneous protein network, resulting from the degradation of the arabinoxylan structure of BSG, and the release of the components entrapped into the cellular compartments. The extensive cell wall disruption contributed to the release of phenols, and conferred enhanced antioxidant activity to the fortified pasta. The persistence of the activity was demonstrated after in vitro-mimicked digestion, evaluating the protective effects of the digested pasta towards induced oxidative stress in Caco-2 cells cultures. The fortified pasta showed a peculiar sensory profile, markedly improved by the pre-treatment, thus confirming the great potential of bioprocessed BSG as health-promoting food ingredient.

DeepFRAP: Fast fluorescence recovery after photobleaching data analysis using deep neural networks.

Conventional analysis of fluorescence recovery after photobleaching (FRAP) data for diffusion coefficient estimation typically involves fitting an analytical or numerical FRAP model to the recovery curve data using non-linear least squares. Depending on the model, this can be time consuming, especially for batch analysis of large numbers of data sets and if multiple initial guesses for the parameter vector are used to ensure convergence. In this work, we develop a completely new approach, DeepFRAP, utilizing machine learning for parameter estimation in FRAP. From a numerical FRAP model developed in previous work, we generate a very large set of simulated recovery curve data with realistic noise levels. The data are used for training different deep neural network regression models for prediction of several parameters, most importantly the diffusion coefficient. The neural networks are extremely fast and can estimate the parameters orders of magnitude faster than least squares. The performance of the neural network estimation framework is compared to conventional least squares estimation on simulated data, and found to be strikingly similar. Also, a simple experimental validation is performed, demonstrating excellent agreement between the two methods. We make the data and code used publicly available to facilitate further development of machine learning-based estimation in FRAP. LAY DESCRIPTION: Fluorescence recovery after photobleaching (FRAP) is one of the most frequently used methods for microscopy-based diffusion measurements and broadly used in materials science, pharmaceutics, food science and cell biology. In a FRAP experiment, a laser is used to photobleach fluorescent particles in a region. By analysing the recovery of the fluorescence intensity due to the diffusion of still fluorescent particles, the diffusion coefficient and other parameters can be estimated. Typically, a confocal laser scanning microscope (CLSM) is used to image the time evolution of the recovery, and a model is fit using least squares to obtain parameter estimates. In this work, we introduce a new, fast and accurate method for analysis of data from FRAP. The new method is based on using artificial neural networks to predict parameter values, such as the diffusion coefficient, effectively circumventing classical least squares fitting. This leads to a dramatic speed-up, especially noticeable when analysing large numbers of FRAP data sets, while still producing results in excellent agreement with least squares. Further, the neural network estimates can be used as very good initial guesses for least squares estimation in order to make the least squares optimization convergence much faster than it otherwise would. This provides for obtaining, for example, diffusion coefficients as soon as possible, spending minimal time on data analysis. In this fashion, the proposed method facilitates efficient use of the experimentalist's time which is the main motivation to our approach. The concept is demonstrated on pure diffusion. However, the concept can easily be extended to the diffusion and binding case. The concept is likely to be useful in all application areas of FRAP, including diffusion in cells, gels and solutions.

Bioprocessing of Brewers' Spent Grain Enhances Its Antioxidant Activity: Characterization of Phenolic Compounds and Bioactive Peptides.

Brewers' spent grain (BSG) is the major by-product of the brewing industry which remain largely unutilized despite its nutritional quality. In this study, the effects of fermentation on BSG antioxidant potential were analyzed. A biotechnological protocol including the use of xylanase followed by fermentation with Lactiplantibacillus plantarum (Lactobacillus plantarum) PU1, PRO17, and H46 was used. Bioprocessed BSG exhibited enhanced antioxidant potential, characterized by high radical scavenging activity, long-term inhibition of linoleic acid oxidation and protective effect toward oxidative stress on human keratinocytes NCTC 2544. Immunolabelling and confocal laser microscopy showed that xylanase caused an extensive cell wall arabinoxylan disruption, contributing to the release of bound phenols molecules, thus available to further conversion through lactic acid bacteria metabolism. To clarify the role of fermentation on the antioxidant BSG potential, phenols were selectively extracted and characterized through HPLC-MS techniques. Novel antioxidant peptides were purified and identified in the most active bioprocessed BSG.

A Highly Accurate Pixel-Based FRAP Model Based on Spectral-Domain Numerical Methods.

We introduce a new, to our knowledge, numerical model based on spectral methods for analysis of fluorescence recovery after photobleaching data. The model covers pure diffusion and diffusion and binding (reaction-diffusion) with immobile binding sites, as well as arbitrary bleach region shapes. Fitting of the model is supported using both conventional recovery-curve-based estimation and pixel-based estimation, in which all individual pixels in the data are utilized. The model explicitly accounts for multiple bleach frames, diffusion (and binding) during bleaching, and bleaching during imaging. To our knowledge, no other fluorescence recovery after photobleaching framework incorporates all these model features and estimation methods. We thoroughly validate the model by comparison to stochastic simulations of particle dynamics and find it to be highly accurate. We perform simulation studies to compare recovery-curve-based estimation and pixel-based estimation in realistic settings and show that pixel-based estimation is the better method for parameter estimation as well as for distinguishing pure diffusion from diffusion and binding. We show that accounting for multiple bleach frames is important and that the effect of neglecting this is qualitatively different for the two estimation methods. We perform a simple experimental validation showing that pixel-based estimation provides better agreement with literature values than recovery-curve-based estimation and that accounting for multiple bleach frames improves the result. Further, the software developed in this work is freely available online.

Increased release of carotenoids and delayed in vitro lipid digestion of high pressure homogenized tomato and pepper emulsions.

Carotenoids are lipophilic compounds that are digested and absorbed along with lipids. Emulsions based on a mixture of plum tomato and red sweet pepper, with 5% or 10% rapeseed oil, were obtained by high pressure homogenization, and the concentration of carotenoids in the emulsion oil droplets was quantified. The fraction of lycopene and beta-carotene released from the plant matrix into the oil droplets was highest in the 10% emulsion, which had larger oil droplets than the 5% emulsion. Xanthophylls were easily released into oil droplets in both 5% and 10% emulsions. The results suggest that the release of carotenoids made available for intestinal absorption depends on carotenoid type and can be significantly improved by increasing the homogenization pressure and oil content. However, in vitro gastrointestinal digestion indicated the presence of constituents or structures in the emulsions, originating from tomato, that reduced pancreatic activity, which may delay micellarization and uptake of carotenoids.

An antibiofilm coating of 5-aryl-2-aminoimidazole covalently attached to a titanium surface.

Biofilms, especially those formed by Staphylococcus aureus, play a key role in the development of orthopedic implant infections. Eradication of these infections is challenging due to the elevated tolerance of biofilm cells against antimicrobial agents. In this study, we developed an antibiofilm coating consisting of 5-(4-bromophenyl)-N-cyclopentyl-1-octyl-1H-imidazol-2-amine, designated as LC0024, covalently bound to a titanium implant surface (LC0024-Ti). We showed in vitro that the LC0024-Ti surface reduces biofilm formation of S. aureus in a specific manner without reducing the planktonic cells above the biofilm, as evaluated by plate counting and fluorescence microscopy. The advantage of compounds that only inhibit biofilm formation without affecting the viability of the planktonic cells, is that reduced development of bacterial resistance is expected. To determine the antibiofilm activity of LC0024-Ti surfaces in vivo, a biomaterial-associated murine infection model was used. The results indicated a significant reduction in S. aureus biofilm formation (up to 96%) on the LC0024-Ti substrates compared to pristine titanium controls. Additionally, we found that the LC0024-Ti substrates did not affect the attachment and proliferation of human cells involved in osseointegration and bone repair. In summary, our results emphasize the clinical potential of covalent coatings of LC0024 on titanium implant surfaces to reduce the risk of orthopedic implant infections. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1908-1919, 2019.

Self-Assembly of Recombinant Silk as a Strategy for Chemical-Free Formation of Bioactive Coatings: A Real-Time Study.

Functionalization of biomaterials with biologically active peptides can improve their performance after implantation. By genetic fusion to self-assembling proteins, the functional peptides can easily be presented on different physical formats. Herein, a chemical-free coating method based on self-assembly of the recombinant spider silk protein 4RepCT is described and used to prepare functional coatings on various biomaterial surfaces. The silk assembly was studied in real-time, revealing the occurrence of continuous assembly of silk proteins onto surfaces and the formation of nanofibrillar structures. The adsorbed amounts and viscoelastic properties were evaluated, and the coatings were shown to be stable against wash with hydrogen chloride, sodium hydroxide, and ethanol. Titanium, stainless steel, and hydroxyapatite were coated with silk fused to an antimicrobial peptide or a motif from fibronectin. Human primary cells cultured on the functional silk coatings show good cell viability and proliferation, implying the potential to improve implant performance and acceptance by the body.

Antibacterial activity of a new broad-spectrum antibiotic covalently bound to titanium surfaces.

Biofilm-associated infections, particularly those caused by Staphylococcus aureus, are a major cause of implant failure. Covalent coupling of broad-spectrum antimicrobials to implants is a promising approach to reduce the risk of infections. In this study, we developed titanium substrates on which the recently discovered antibacterial agent SPI031, a N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol, was covalently linked (SPI031-Ti). We found that SPI031-Ti substrates prevent biofilm formation of S. aureus and Pseudomonas aeruginosa in vitro, as quantified by plate counting and fluorescence microscopy. To test the effectiveness of SPI031-Ti substrates in vivo, we used an adapted in vivo biomaterial-associated infection model in mice in which SPI031-Ti substrates were implanted subcutaneously and subsequently inoculated with S. aureus. Using this model, we found a significant reduction in biofilm formation (up to 98%) on SPI031-Ti substrates compared to control substrates. Finally, we demonstrated that the functionalization of the titanium surfaces with SPI031 did not influence the adhesion and proliferation of human cells important for osseointegration and bone repair. In conclusion, these data demonstrate the clinical potential of SPI031 to be used as an antibacterial coating for implants, thereby reducing the incidence of implant-associated infections. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2191-2198, 2016.

Covalent immobilization of antimicrobial agents on titanium prevents Staphylococcus aureus and Candida albicans colonization and biofilm formation.

OBJECTIVES: Biofilm-associated implant infections represent a serious public health problem. Covalent immobilization of antimicrobial agents on titanium (Ti), thereby inhibiting biofilm formation of microbial pathogens, is a solution to this problem. METHODS: Vancomycin (VAN) and caspofungin (CAS) were covalently bound on Ti substrates using an improved processing technique adapted to large-scale coating of implants. Resistance of the VAN-coated Ti (VAN-Ti) and CAS-coated Ti (CAS-Ti) substrates against in vitro biofilm formation of the bacterium Staphylococcus aureus and the fungal pathogen Candida albicans was determined by plate counting and visualized by confocal laser scanning microscopy. The efficacy of the coated Ti substrates was also tested in vivo using an adapted biomaterial-associated murine infection model in which control-Ti, VAN-Ti or CAS-Ti substrates were implanted subcutaneously and subsequently challenged with the respective pathogens. The osseointegration potential of VAN-Ti and CAS-Ti was examined in vitro using human bone marrow-derived stromal cells, and for VAN-Ti also in a rat osseointegration model. RESULTS: In vitro biofilm formation of S. aureus and C. albicans on VAN-Ti and CAS-Ti substrates, respectively, was significantly reduced compared with biofilm formation on control-Ti. In vivo, we observed over 99.9% reduction in biofilm formation of S. aureus on VAN-Ti substrates and 89% reduction in biofilm formation of C. albicans on CAS-Ti substrates, compared with control-Ti substrates. The coated substrates supported osseointegration in vitro and in vivo. CONCLUSIONS: These data demonstrate the clinical potential of covalently bound VAN and CAS on Ti to reduce microbial biofilm formation without jeopardizing osseointegration.

Identification and characterization of an anti-pseudomonal dichlorocarbazol derivative displaying anti-biofilm activity.

Pseudomonas aeruginosa strains resistant towards all currently available antibiotics are increasingly encountered, raising the need for new anti-pseudomonal drugs. We therefore conducted a medium-throughput screen of a small-molecule collection resulting in the identification of the N-alkylated 3,6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol (MIC = 18.5 µg mL⁻¹). This compound, compound 1, is bacteriostatic towards a broad spectrum of Gram-positive and Gram-negative pathogens, including P. aeruginosa. Importantly, 1 also eradicates mature biofilms of P. aeruginosa. 1 displays no cytotoxicity against various human cell types, pointing to its potential for further development as a novel antibacterial drug.

Changes in salt solubility and microstructure of proteins from herring (Clupea harengus) after pH-shift processing.

Salt solubility of pH-shift isolated herring (Clupea harengus) muscle proteins was studied in relation to pH exposure and microstructure using transmission electron microscopy (TEM). Using protein solubilization at pH 11.2 with subsequent precipitation at pH 5.5, salt solubility of the proteins decreased from 78 to 17%. By precipitating the alkali-solubilized proteins at the pH of native herring muscle, 6.5, salt solubility only decreased to 59%, proving that pH values between 6.5 and 5.5 affected protein salt solubility more than the pH cycle 6.5 → 11.2 → 6.5. Precipitation at pH 5.5 resulted in hydrogen bonds, hydrophobic interactions, and S-S bridges, whereas precipitation at pH 6.5 resulted only in the formation of hydrophobic interactions. The alkaline pH-shift isolation process severely rearranged the protein microstructure, with precipitation at pH 6.5 forming a finer, more homogeneous network than precipitation at pH 5.5. The former protein isolate also contained less lipid oxidation products and formed more deformable gels, without affecting protein yield.

Oncostatin M-induced genes in human astrocytomas.

Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) cytokine family and known to be induced in the nervous system as a result of cell stress. OSM is expressed in most human brain tumors, but the effects on tumor cells are unclear. The cytokine is known to activate the JAK/STAT signaling pathway by binding to its receptors gp130/OSMbeta or gp130/LIFRbeta and thereby initiating activation or suppression of a number of STAT target genes. The objective of the study was to identify OSM-regulated genes that could help in understanding the function of OSM in glioma cells. The glioma cell line, U1242MG was stimulated by OSM and the gene expression patterns were analyzed by microarray. In total, nineteen differentially expressed genes were selected due to high intensity, level of up/down-regulation and biological functions. The differentially expressed genes were verified using quantitative PCR. Additional validation of the confirmed OSM-induced proteins was performed in human astrocytoma tissues by immunohistochemistry. Among the up-regulated genes were CHI3L1, PLAU, MT2A and EPAS1. These genes are known to be involved in cell matrix remodeling, migration, proliferation control and angiogenesis. The results suggest that OSM induces genes that might contribute to the development and progression of astrocytomas.

Oncostatin M signaling in human glioma cell lines.

We have recently found that oncostatin M (OSM) is overexpressed in most human brain tumors. The effects of OSM are unclear with conflicting reports of growth stimulatory or inhibitory effects in various cell types. The aim of this study was to investigate the effects of OSM in 5 glioma cell lines and 7 short-term cultures of human gliomas and in normal cultured human astrocytes. None of the cell lines and short-term cultured tumor cells expressed OSM in vitro. OSM signals through a gp130 containing receptor complex over the JAK/STAT pathway. Immunofluorescence and RT-PCR analysis showed that the tumor cells express gp130 and the other receptor components, LIFRbeta and OSMRbeta. OSM treatment induced phosphorylation of STAT3 and STAT1 indicating presence of a functional JAK/STAT pathway. No OSM effect on proliferation was observed. OSM gave no protective effects against tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced cytotoxicity.