The red macroalgae Porphyra, commonly known as Nori, is widely used as food around the world due to its high nutrient content, including the significant abundance of colored phycobiliproteins (PBPs). Among these, R-phycocyanin (R-PC) stands out for its vibrant purple color and numerous bioactive properties, making it a valuable protein for the food industry. However, R-PC\'s limited thermal stability necessitates alternative processing methods to preserve its color and bioactive properties. Our study aimed to investigate the in-situ stability of oligomeric R-PC under high pressure (HP) conditions (up to 4000 bar) using a combination of absorption, fluorescence, and small-angle X-ray scattering (SAXS) techniques. The unfolding of R-PC is a multiphase process. Initially, low pressure induces conformational changes in the R-PC oligomeric form (trimers). As pressure increases above 1600 bar, these trimers dissociate into monomers, and at pressures above 3000 bar, the subunits begin to unfold. When returned to atmospheric pressure, R-PC partially refolds, retaining 50% of its original color absorbance. In contrast, heat treatment causes irreversible and detrimental effects on R-PC color, highlighting the advantages of HP treatment in preserving both the color and bioactive properties of R-PC compared to heat treatment.

Driven by the acknowledged health and functional properties of milk fat globules (MFGs), there is a growing interest to develop gentle methodologies for separation of fat from milk. In this study, separation of fat from raw milk and fractionation in streams containing MFGs of different size was achieved using a series of two silicon carbide ceramic membranes. A first step consisting of a 1.4 µm membrane aimed to concentrate the bulk of the fat, i.e. the larger MFGs (D[4,3] ∼ 4 µm) followed by a 0.5 µm fractionation aimed to concentrate the residual milk fat in the permeate, i.e. fraction with the smaller MFGs (D[4,3] ∼ 1.8-2.4 µm. The fat separation performance showed a yield of 92 % for the 1.4 µm membrane and 97 % for the 0.5 µm membrane. Both fat enriched retentates showed, by the confocal laser scanning microscopy, intact MFGs with limited damage in the MFG membrane. The fatty acid profile analysis and SAXS showed minor differences in fat acid composition and the crystallization behavior was related to differences in the fat content. The 0.5 µm permeate containing the smallest MFGs however showed larger aggregates and a trinomial particle size distribution, due to probably pore pressure induced coalescences. The series of silicon carbide membranes showed potential to concentrate some of MFGM proteins such as Periodic Schiff base 3/4 and cluster of differentiation 36 especially in the 0.5 µm retentates. A shift in casein to whey protein ratio from 80:20 (milk) to 50:50 was obtained in the final 0.5 µm permeate, which opens new opportunities for product development.

A novel smart film MP/BNC/ACN for real-time monitoring of fish freshness was developed using myofibrillar protein (MP) and bacterial nanocellulose (BNC) as film raw materials and anthocyanin (Lycium ruthenicum, ACN) as an indicator. Firstly, the film containing 1 % ACN (MP/BNC/ACN1) was found to have a moderate thickness (0.44 ± 0.01 mm) and superior mechanical properties (tensile strength (TS) = 8.53 ± 0.11 MPa; elongation at break (EB) = 24.85 ± 1.38 %) by determining the physical structure. The covalent, electrostatic, and hydrogen bonding interactions between anthocyanin and the film matrix were identified and confirmed by FT-IR spectroscopy (FTIR), X-ray diffraction (XRD), and scanning electron microscope (SEM) analysis. A comprehensive evaluation concluded that MP/BNC/ACN1 exhibited excellent trimethylamine (TMA) sensitivity (total color difference (ΔE), ΔETMA0-1000 = 4.47-31.05; limit of detection (LOD), LOD = 1.03) and UV stability (ΔE96h = 4.16 ± 0.13). The performance of the films in assessing fish freshness was evaluated, principal component analysis (PCA) and hierarchical cluster analysis (HCA) revealed that MP/BNC/ACN1 (ΔE2-10d = 16.84-32.05) could clearly distinguish between fresh (0-2 d), sub-fresh (4-6 d), and spoiled (8-10 d) stages of fish, which corresponded to the film colors of red, light red, and gray-black. In conclusion, this study addresses the limitation that intelligent films cannot visually discern real-time freshness during fish storage and provides a promising approach for real-time fish freshness monitoring.

Iron insertion into porphyrins is an essential step in heme biosynthesis. In the coproporphyrin-dependent pathway, specific to monoderm bacteria, this reaction is catalyzed by the monomeric enzyme coproporphyrin ferrochelatase. In addition to the mechanistic details of the metalation of the porphyrin, the identification of the substrate access channel for ferrous iron to the active site is important to fully understand this enzymatic system. In fact, whether the iron reaches the active site from the distal or the proximal porphyrin side is still under debate. In this study we have thoroughly addressed this question in Listeria monocytogenes coproporphyrin ferrochelatase by X-ray crystallography, steady-state and pre-steady-state imidazole ligand binding studies, together with a detailed spectroscopic characterization using resonance Raman and UV-vis absorption spectroscopies in solution. Analysis of the X-ray structures of coproporphyrin ferrochelatase-coproporphyrin III crystals soaked with ferrous iron shows that iron is present on both sides of the porphyrin. The kinetic and spectroscopic study of imidazole binding to coproporphyrin ferrochelatase‑iron coproporphyrin III clearly indicates the presence of two possible binding sites in this monomeric enzyme that influence each other, which is confirmed by the observed cooperativity at steady-state and a biphasic behavior in the pre-steady-state experiments. The current results are discussed in the context of the entire heme biosynthetic pathway and pave the way for future studies focusing on protein-protein interactions.

Spatial hindrance-based pro-antibodies (pro-Abs) are engineered antibodies to reduce monoclonal antibodies\' (mAbs) on-target toxicity using universal designed blocking segments that mask mAb antigen-binding sites through spatial hindrance. By linking through protease substrates and linkers, these blocking segments can be removed site-specifically. Although many types of blocking segments have been developed, such as coiled-coil and hinge-based Ab locks, the molecular structure of the pro-Ab, particularly the region showing how the blocking fragment blocks the mAb, has not been elucidated by X-ray crystallography or cryo-EM. To achieve maximal effect, a pro-Ab must have high antigen-blocking and protease-restoring efficiencies, but the unclear structure limits its further optimization. Here, we utilized molecular dynamics (MD) simulations to study the dynamic structures of a hinge-based Ab lock pro-Ab, pro-Nivolumab, and validated the simulated structures with small- and wide-angle X-ray scattering (SWAXS). The MD results were closely consistent with SWAXS data (χ2 best-fit = 1.845, χ2 allMD = 3.080). The further analysis shows a pronounced flexibility of the Ab lock (root-mean-square deviation = 10.90 Å), yet it still masks the important antigen-binding residues by 57.3%-88.4%, explaining its 250-folded antigen-blocking efficiency. The introduced protease accessible surface area method affirmed better protease efficiency for light chain (33.03 Å2) over heavy chain (5.06 Å2), which aligns with the experiments. Overall, we developed MD-SWAXS validation method to study the dynamics of flexible blocking segments and introduced methodologies to estimate their antigen-blocking and protease-restoring efficiencies, which would potentially be advancing the clinical applications of any spatial hindrance-based pro-Ab.

The GdAl3(BO3)4:xPr3+ (0 ≤ x ≤ 5.0 mol%) phosphors were prepared through solid state reaction route and characterized for various lighting applications. Powder X-ray diffraction investigations revel rhombohedral structure matched to JCPDS card no. 83-1907. The morphological studies confirm the agglomeration of particles with different size and shape. The emission spectra show various emission transitions originating from Pr3+:(3P1,0, 1D2) emission states to their lower lying energy states upon 274 nm NUV excitation with a red shift for x > 0.5 mol%. The colour perception analysis results an intense red luminescence due to efficient energy transfer from Gd3+ to Pr3+ ions. The temperature-dependent luminescence investigations show good thermal stability even beyond 150°C with an activation energy of 0.24 eV. The observed experimental results show the potentiality of GdAl3(BO3)4:0.5 Pr3+ phosphor for red emitting devices and red component in phosphor converted white LEDs.

The characteristics of dopamine self-polymerization were used to cover the nano-titanium dioxide (TiO2) surface and produce nano-titanium dioxide-polydopamine (TiO2-PDA). The reducing nature of dopamine was then used to reduce silver nitrate to silver elemental particles on the modified nano-titanium dioxide: The resulting TiO2-PDA-Ag nanoparticles were used as antimicrobial agents. Finally, the antibacterial agent was mixed with silicone to obtain an antibacterial silicone composite material. The composition and structure of antibacterial agents were analyzed by scanning electron microscopy, transmission electron microscopy, X-ray photoelectron energy spectroscopy, and X-ray diffraction. Microscopy and the antibacterial properties of the silicone antibacterial composites were studied as well. The TiO2-PDA-Ag antimicrobial agent had good dispersion versus nano-TiO2. The three were strongly combined with obvious characteristic peaks. The antibacterial agents were evenly dispersed in silicone, and the silicone composite has excellent antibacterial properties. Bacillus subtilis (B. subtilis) adhesion was reduced from 246 × 104 cfu/cm2 to 2 × 104 cfu/cm2, and colibacillus (E. coli) reduced from 228 × 104 cfu/cm2 leading to bacteria-free adhesion.

The dissolution and bioavailability challenges posed by poorly water-soluble drugs continue to drive innovation in pharmaceutical formulation design. Nintedanib (NDNB) is a typical BCS class II drug that has been utilized to treat idiopathic pulmonary fibrosis (IPF). Due to the low solubility, its oral bioavailability is relatively low, limiting its therapeutical effectiveness. It is crucial to enhance the dissolution and the oral bioavailability of NDNB. In this study, we focused on the preparation of amorphous solid dispersions (ASD) using hot melt extrusion (HME). The formulation employed Kollidon® VA64 (VA64) as the polymer matrix, blended with the NDNB at a ratio of 9:1. HME was conducted at temperatures ranging from 80 °C to 220 °C. The successful preparation of ASD was confirmed through various tests including polarized light microscopy (PLM), X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FT-IR), and thermogravimetric analysis (TGA). The in-vitro cumulative release of NDNB-ASD in 2 h in a pH 6.8 medium was 8.3-fold higher than that of NDNB (p < 0.0001). In a pH 7.4 medium, it was 10 times higher (p < 0.0001). In the in-vivo pharmacokinetic experiments, the area under curve (AUC) of NDNB-ASD was 5.3-fold higher than that of NDNB and 2.2 times higher than that of commercially available soft capsules (Ofev®) (p < 0.0001). There was no recrystallization after 6 months under accelarated storage test. Our study indicated that NDNB-ASD can enhance the absorption of NDNB, thus providing a promising method to improve NDNB bioavailability in oral dosages.

Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction of humans, domestic and research animals, and endangered species using cryopreserved oocytes and embryos. The nature of ice formed in bovine oocytes (similar in size to oocytes of humans and most other mammals) after rapid cooling and during rapid warming was examined using synchrotron-based time-resolved x-ray diffraction. Using cooling rates, warming rates and CPA concentrations of current practice, oocytes show no ice after cooling but always develop large ice fractions-consistent with crystallization of most free water-during warming, so most ice-related damage must occur during warming. The detailed behavior of ice at warming depended on the nature of ice formed during cooling. Increasing cooling rates allows oocytes soaked as in current practice to remain essentially ice free during both cooling and warming. Much larger convective warming rates are demonstrated and will allow routine ice-free cryopreservation with smaller CPA concentrations. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes and establish the structure and grain size of ice formed. Ice formation can be eliminated as a factor affecting post-warming oocyte viability and development in many species, improving outcomes and allowing other deleterious effects of the cryopreservation cycle to be independently studied.

OBJECTIVE: This study evaluated the phase composition, phase transformation behaviour, and mechanical properties of five heat-treated NiTi instruments.
METHODS: ProTaper NEXT (M-wire, PTN), ProTaper Gold (Gold-wire, PTG), One Curve (C-wire, OC), EdgeTaper Platinum (Fire-wire, ETP), NeoNiTi (electrical discharge machining-wire, NNA), and ProTaper Universal (conventional wire, PTU, control) with #25-tip size were tested (n = 12/group) for cyclic fatigue resistance (number of cycles to failure; NCF) and torsional resistance (angle of rotation to fracture and maximum torque at fracture [ultimate torsional strength]). The geometry and fracture surfaces of the tested instruments were examined by scanning electron microscopy. The phase transformation temperature and phase composition of the instruments were evaluated using differential scanning calorimetry and X-ray diffraction. Data were statistically analysed using one-way ANOVA and Tukey\'s post hoc test, with the significance level set at 5%.
RESULTS: PTG showed the highest NCF (P < .05) at 37°C, while ETP exhibited the highest angle of rotation to fracture, ultimate torsional strength, and stiffness (P < .05). Scanning electron microscopy demonstrated typical clusters of fatigue striations and numerous cracks after cyclic fatigue fracture, whereas there was a concentric abrasion pattern with a dimple and microvoids at the centre after torsional fracture. In differential scanning calorimetry curves, austenite-finishing temperatures of heat-treated instruments were higher than 37°C, whereas that of PTU was lower than 37°C. PTU showed strong peaks of austenite at 25 and 37°C, whereas ETP showed a strong peak of R-phase at 25°C, but mostly austenite phase at 37°C in X-ray diffraction.
CONCLUSIONS: Geometry, alloy type, and phase transformation temperatures of NiTi instruments affected their mechanical behaviour.
CONCLUSIONS: PTG showed the highest NCF, suitable for markedly curved canals. ETP had the highest torsional resistance, appropriate for narrow and constricted canals.