Diphenyl-Methane Based Thyromimetic Inhibitors for Transthyretin Amyloidosis

Thyromimetics, whose physicochemical characteristics are analog to thyroid hormones (THs) and their derivatives, are promising candidates as novel therapeutics for neurodegenerative and metabolic pathologies. In particular, sobetirome (GC-1), one of the initial halogen-free thyromimetics, and newly synthesized IS25 and TG68, with optimized ADME-Tox profile, have recently attracted attention owing to their superior therapeutic benefits, selectivity, and enhanced permeability. Here, we further explored the functional capabilities of these thyromimetics to inhibit transthyretin (TTR) amyloidosis. TTR is a homotetrameric transporter protein for THs, yet it is also responsible for severe amyloid fibril formation, which is facilitated by tetramer dissociation into non-native monomers. By combining nuclear magnetic resonance (NMR) spectroscopy, computational simulation, and biochemical assays, we found that GC-1 and newly designed diphenyl-methane-based thyromimetics, namely IS25 and TG68, are TTR stabilizers and efficient suppressors of TTR aggregation. Based on these observations, we propose the novel potential of thyromimetics as a multi-functional therapeutic molecule for TTR-related pathologies, including neurodegenerative diseases.     

Production of the Carboxylate Reductase from Nocardia otitidiscaviarum in a Soluble,Active Form for in vitro Applications

Accessing aldehydes from carboxylate moieties is often a challenging task. In this regard, carboxylate reductases (CARs) are promising catalysts provided by nature that are able to accomplish this task in just one step, avoiding over-reduction to the alcohol product. However, the heterologous expression of CARs can be quite difficult due to the excessive formation of insoluble protein, thus hindering further characterization and application of the enzyme. Here, the heterologous production of the carboxylate reductase from Nocardia otitidiscaviarum (NoCAR) was optimized by a combination of i) optimized cultivation conditions, ii) post-translational modification with a phosphopantetheinyl transferase and iii) selection of an appropriate expression strain. Especially, the selection of Escherichia coli tuner cells as host had a strong effect on the final 110-fold increase in the specific activity of NoCAR. This highly active NoCAR was used to reduce sodium benzoate to benzaldehyde, and it was successfully assembled with an in vitro regeneration of ATP and NADPH, being capable of reducing about 30 mM sodium benzoate with high selectivity in only 2 h of reaction.     

Alpha Synuclein only Forms Fibrils In Vitro when Larger than its Critical Size of 70 Monomers

The aggregation of α-synuclein into small soluble aggregates and then fibrils is important in the development and spreading of aggregates through the brain in Parkinson's disease. Fibrillar aggregates can grow by monomer addition and then break into fragments that could spread into neighboring cells. The rate constants for fibril elongation and fragmentation have been measured but it is not known how large an aggregate needs to be before fibril formation is thermodynamically favorable. This critical size is an important parameter controlling at what stage in an aggregation reaction fibrils can form and replicate. We determined this value to be approximately 70 monomers using super-resolution and atomic force microscopy imaging of individual α-synuclein aggregates formed in solution over long time periods. This represents the minimum size for a stable α-synuclein fibril and we hypothesis the formation of aggregates of this size in a cell represents a tipping point at which rapid replication occurs.     

Development of protease nanobiocatalysts and their application in hydrolysis of sunflower meal protein isolate

In this study, the suitability of fumed silica nanoparticles (FNS) and its derivatives (amino-modified FNS (AFNS), cyanuric chloride-activated AFNS (CCAFNS) and epoxy-modified FNS (GFNS)), for covalent immobilisation of two commercial protease preparations Alcalase^® and Flavourzyme^® was investigated. The highest hydrolytic activities of immobilised preparations were 25 IU g⁻¹ support (Alcalase-GFNS) and 2.95 IU g⁻¹ support (Flavourzyme-CCAFNS). Furthermore, the immobilised preparations showed 43% and 20% of initial specific activities of commercial protease preparations, respectively. Flavourzyme-CCAFNS also exhibited the highest exopeptidase activity of 22.83 L-pNAU g⁻¹ support. Finally, these two nanobiocatalysts were successfully applied for hydrolysis of sunflower meal protein isolate (SMPI), providing two times higher hydrolysis yields in comparison to free enzymes, justifying the applied immobilisation process. Namely, the highest hydrolysis yield (30%) was gained by the sequential hydrolysis with Alcalase-GFNS and Flavourzyme-CCAFNS, which resulted in the formation of small hydrophobic and hydrophilic peptides, ≤5 kDa, confirmed by HPLC analysis and electrophoretic separation.     

Weissella cibaria short-fermented liquid sourdoughs based on quinoa or amaranth flours as fat replacer in focaccia bread formulation

Production of the traditional Italian Focaccia bread (FcBread) requires reformulation to meet the new rules for healthier foods. We tested the applicability of a short-fermented (15 h) liquid sourdough (LS) (dough yield, DY, 250), obtained using quinoa (Q) or amaranth (Am) flour fermented with a Weissella cibaria strain (C43-11) producing exopolysaccharides (EPS) as fat replacer in yeast-leavened FcBread. The LSs were applied during the FcBread-making process at industrial pilot plant, reducing the added fat amount by 20% (FcBread-QLS and FcBread-AmLS) and products were compared with reference samples not containing EPS. All the products were analysed for physico-chemical properties (pH, TTA, organic acids, protein content and profile, instrumental colour), textural properties, sensory quality and glycemic index. The application of both pseudo-cereal-based W. cibaria LSs in FcBread allowed obtaining products with a reduced amount of fat, increased protein amount, mainly due to the albumin and glutenin/glutelin fractions, reduced glycemic index, improved texture and preserved in the traditional sensory profile of ‘focaccia’.     

Glycation of soy protein isolate with two ketoses: d-Allulose and fructose

Modification of food proteins to have improved functional properties is of great importance. In this study, modification of soy protein isolate (SPI) was achieved through glycation. SPI was glycated in a spray dryer (SD) and an incubator followed by freeze drying (FD). d -Allulose, an important rare sugar, was used in SPI glycation as the carbohydrate source, and results were compared with fructose. In addition to the sugar type, two different SPI powder: sugar ratios (1:1 and 5:1) were investigated. For the glycated samples, emulsification activity, free amino groups, protein solubility, Fourier-transform infrared spectroscopy analysis, antioxidant activity experiments and time-domain NMR relaxometry measurements for hydration were conducted. According to the results, the solubility of SPI that is limited in native form has shown a significant improvement after glycation through both FD and SD methods. Besides, glycation through the FD method was found to be more favourable due to its milder conditions than the SD method. Considering the physicochemical properties, the best combination for the highest glycation degree was found to be the samples prepared at the 1:1 ratio with d -Allulose in the FD method. Overall, it was concluded that glycation of SPI enhanced its functional properties such as antioxidant and emulsification activities.     

Evaluation of antibacterial protein with antioxidant activity from Rahnella aquatilis L103 and its effect on beef during refrigerated storage

A strain, Rahnella aquatilis L103, which was isolated from the soil around the roots of mushrooms, produced antibacterial protein though fermentation. This protein has broad antibacterial spectrum towards gram-positive and gram-negative bacteria. The protein has a significant antioxidant capacity on scavenging ABTS^+· (82.5%, 120 μg mL⁻¹), DPPH^· (64.1%, 600 μg mL⁻¹) and OH^· (60.1%, 750 μg mL⁻¹) as well. The protein was considered to be safe within concentration of 150 μg mL⁻¹ as shown by MTT results and was applied to beef refrigerated preservation. The treatment group presented lower total viable counts (TVC), total volatile basic nitrogen (TVB-N), 2-thiobarbituric acid (TBA), meanwhile, higher sensory score and longer shelf life (2 days) compared with the control group (P < 0.05). The results indicated that the protein under safe concentration was beneficial to beef preservation.     

Structures and properties of chicken myofibrillar protein gel induced by microwave heating

Structures and properties of myofibrillar protein gel prepared at different power (300–800 W) were evaluated. Amino acid analysis demonstrated that changes in microwave power did not alter primary structure of gel. However, an increase in microwave power could change higher structures of gel. As microwave power increased, α-helix content decreased and β-sheet content increased. Increased microwave power probably facilitated protein to unfold and expose the internal groups, causing surface hydrophobicity and the formation of disulphide bonds were enhanced, which indicated changes in tertiary and quaternary structures of protein. At 500 W, gel had the best ultrastructure where surface morphology, springiness and water holding capacity reached the optimum. Our findings suggested that microwave at an appropriate power (500 W) could change higher structures of myofibrillar protein gel to achieve desired processing and quality protein gel characteristics.     

Effects of malondialdehyde-induced protein oxidation on the structural characteristics of rice protein

Malondialdehyde (MDA) was selected to represent a secondary by-product of lipid peroxidation during rice ageing. This study aimed to investigate the effects of MDA modification on the structural characteristics of rice protein. The results showed that as MDA concentration increased, rice protein carbonyl and disulphide groups increased, but sulphydryl content decreased. The blue shift of maximum fluorescence peak, the decrease of rice protein intrinsic fluorescence intensity and the reduction of surface hydrophobicity indicated the formation of protein aggregates caused by MDA oxidative modification. The results of molecular weight distribution and particle size distribution showed that MDA modification resulted in the formation of soluble protein aggregates, and the decrease of rice protein solubility indicated that insoluble protein aggregates were formed. Results of protein electrophoresis showed that MDA modification contributed to rice protein aggregation via non-disulphide covalent bonds. The results showed that rice protein gradually aggregated with increasing MDA concentration.     

Insights into Protein Stability in Cell Lysate by 19F NMR Spectroscopy

In living organisms, protein folding and function take place in an inhomogeneous, highly crowded environment possessing a concentration of diverse macromolecules of up to 400 g/L. It has been shown that the intracellular environment has a pronounced effect on the stability, dynamics and function of the protein under study, and has for this reason to be considered. However, most protein studies neglect the presence of these macromolecules. Consequently, we probe here the overall thermodynamic stability of cold shock protein B from Bacillus subtilis (BsCspB) in cell lysate. We found that an increase in cell lysate concentration causes a monotonic increase in the thermodynamic stability of BsCspB. This result strongly underlines the importance of considering the biological environment when inherent protein parameters are quantitatively determined. Moreover, we demonstrate that targeted application of ¹⁹F NMR spectroscopy operates as an ideal tool for protein studies performed in complex cellular surroundings.