Search Results (154)

Sperm cryo-tolerance resulted in significant variations in post-thaw semen quality among breeds and individual boars. In the present study, semen samples from thirty-seven large white boars were cryopreserved to select individuals with strong and weak freezing tolerance according to their post-thaw sperm quality. Comparative TMT-based quantitative proteomic analysis between the two groups identified 22 significantly differentially expressed proteins. NT5C1B and ADA, the significantly downregulated proteins in the semen of the low cryo-tolerance group, were supplemented in the semen samples with lower cryo-tolerance. Supplementation with 1 µg/mL of NT5C1B dramatically (p < 0.05) improved kinematic parameters and structural integrity. In comparison with the control group, mitochondrial activity and antioxidant capacity were significantly enhanced in post-thaw sperm. In vitro fertilization assays revealed that the NT5C1B-treated group also has notably (p < 0.05) high sperm penetration and embryonic cleavage rates. ADA supplementation did not exhibit obvious freezing tolerance effects. NT5C1B can be a potential key functional protein to enhance the quality and cryo-tolerance during cryopreservation. Specifically, supplementation with 1 µg/mL NT5C1B significantly improved post-thaw motility, structural integrity, mitochondrial activity, and antioxidant capacity and ultimately enhanced the sperm penetration rate and embryonic cleavage rate in cryo-sensitive sperm, confirming its role as a functional protector during cryopreservation. Full article

Cryopreservation is a crucial technique for the long-term preservation of swine genetic resources. However, its efficiency remains limited by cryo-induced oxidative stress, which compromises sperm membrane integrity, mitochondrial function, and fertilizing capacity. Methyl gallate (MG), a naturally occurring polyphenolic antioxidant, has demonstrated strong free radical scavenging and lipid peroxidation inhibitory properties. This study aimed to evaluate the effects of MG supplementation on sperm quality and fertilization capacity during boar semen cryopreservation. Semen samples were cryopreserved in extenders containing different concentrations of MG (0, 10, 20, 30, and 50 µM). Post-thaw sperm quality, oxidative status, mitochondrial activity, apoptosis-related markers, and in vitro fertilization (IVF) outcomes were comprehensively assessed. The results showed that supplementation with 20 µM MG significantly improved post-thaw motility, viability, membrane and acrosome integrity, mitochondrial membrane potential, ATP content, and antioxidant capacity, while decreasing reactive oxygen species (ROS) and malondialdehyde (MDA) levels and reducing apoptosis (p < 0.05). Moreover, the expression of the anti-apoptotic protein BCL-2 was upregulated, whereas that of the pro-apoptotic protein BAX was downregulated. Sperm cryopreserved with 20 µM MG also exhibited a significantly higher IVF cleavage rate compared with the control group (p < 0.05). In conclusion, MG supplementation effectively enhanced boar sperm cryosurvival by maintaining membrane stability, improving mitochondrial function, and mitigating oxidative stress during freezing and thawing. These findings suggest that MG is a promising antioxidant additive for improving the efficiency of boar semen cryopreservation systems. Full article

Bioprinting of Advanced Therapy Medicinal Products offers promising new strategies for personalized medicine, but it requires comprehensive, non-destructive characterization and quality monitoring. To support patients with tailor-made constructs composed of hydrogels and cells derived from allogeneic donors or autologous samples, several challenges must be addressed—such as on-demand production, robust manufacturing, appropriate storage and logistics, and destruction-free quality control—before successful translation into clinical applications or pharmacy is possible. Although experience in cryo-preservation, blood banking, and organ donation helps to identify critical process parameters, detecting variations in manufacturing and ensuring product stability remain essential. Quality monitoring of 3D-printed objects before and after storage by magnetic resonance imaging (MRI) is complemented here by measurements of total mass and volume. These established methods provide rapid, non-destructive feedback and have well-characterized statistical limitations. Total mass can be assessed quickly; however, such integral measurements do not reveal information about internal structures. MRI, in contrast, offers detailed, spatially resolved insights. By combining these analytical modalities, we quantitatively analyzed the storage stability of 3D-printed hydrogels—without living cells in this study—in order to demonstrate and validate the analytical approach. We describe a workflow for measuring mass and geometry of 3D-printed hydrogel lattices before and after storage under varying process parameters. Critical quality attributes (cQAs), including overall and internal structural fidelity as well as mass conservation, were monitored. The presented workflow supports the development of cryopreservation protocols and has potential applications in biomaterial development for bioprinting and in quality assessment of tailor-made artificial tissues. Full article

This study investigated the effects of different microencapsulation wall materials on the physicochemical, textural, thermal, and microstructural properties of probiotic ice cream during frozen storage. Lactobacillus acidophilus ATCC 4356 was encapsulated using lyophilization with whole milk powder, skim milk powder, whey powder, or sorbitol, and added to the ice cream mix at 1% (w/w). Five formulations were produced (control and four encapsulated variants) and analyzed over 90–150 days of storage at −18 °C. The highest firmness (41.96 g) and consistency (58.65 g·s) values were observed in the skim milk powder group, whereas sorbitol decreased viscosity and increased overrun. Melting resistance improved during storage, particularly in skim milk powder samples, where the complete melting time increased to 87.35 min. DSC results showed significantly higher enthalpy in whey powder samples, while sorbitol reduced ice crystal growth. Cryo-SEM images confirmed smoother, denser microstructures in formulations with milk powders and sorbitol. Encapsulation markedly enhanced probiotic survival: while the control decreased from 5.04 to 2.18 log CFU/g, encapsulated samples maintained counts above the therapeutic threshold (≥6 log CFU/g) up to 150 days, with the highest viability in whole and skim milk powder. Overall, milk-based encapsulation systems provided both cryoprotection and quality enhancement, demonstrating that microencapsulation is an effective strategy to produce stable probiotic ice creams with improved structural and technological attributes. Full article

Ti-6Al-4V is valued for its strength-to-weight ratio in engineering applications. Cryo-rolling at sub-zero temperatures enhances strength and hardness through grain refinement and dislocation build-up. The present study investigates the role of cryo-rolling on the microstructural characteristics and mechanical properties of the alloy, which undergoes various degrees of deformation followed by heating at 900 °C for selected samples. Microstructural analysis reveals grain elongation, sub-grain formation, deformation bands, and dislocation densification with increasing thickness reduction. Twinning dominates deformation at low strain, while dislocation slips take over at high strain because of the decrease in grain size, which makes the formation of new twins progressively more challenging. No metastable phase appears during cryo-rolling or heat treatment, as confirmed by X-ray diffraction. Cryo-rolled samples exhibit about 45% and 29% reduction in grain size and crystallite size, and 160% intensification in dislocation density. This leads to rises of 19%, 23%, and 10% in yield strength, tensile strength, and hardness, respectively, while ductility remains nearly constant across all cryo-rolled conditions. Cryo-rolling inhibits dynamic recovery and recrystallisation, so strengthening mainly results from grain refinement and dislocation accumulation. These findings suggest that cryo-rolling can improve the strength and hardness of Ti-6Al-4V, while maintaining ductility and providing new processing insights. Full article

Early life nutrition is a critical factor influencing subsequent performance and quality, including skeletal development, in farmed Atlantic cod (Gadus morhua). This study investigated the effects of a novel start-feed protocol utilizing barnacle nauplii and plankton eggs and two experimental microdiets on larval survival, growth, skeletal anomalies, and organ ontogeny. Atlantic cod larvae were reared using three feeding protocols (COM, D1, and D2): COM used enriched rotifers and a commercial microdiet, while D1 and D2 protocols incorporated blue mussel eggs (Cryo-µ) and barnacle nauplii (Cryo-S, Cryo-L), followed by inert microdiets that differed in their phospholipid (PL) source (D1 richer in vegetable PL; D2 richer in marine PL). Larvae were sampled up to 66 days post hatching (dph) for morphometric, skeletal anomaly, and histological analyses. Survival averaged 21.3% and was unaffected by the diets. The control group had slightly higher standard length and dry weight at 66 dph compared to the experimental groups. However, larvae fed the D1 protocol exhibited a significantly lower overall prevalence of skeletal anomalies (52%) compared to the control group (91%). Moreover, D1 showed a lower occurrence of severe anomalies and a significantly reduced prevalence of scoliosis compared to both D2 and COM groups. Histology showed that group D1 achieved an overall accelerated organ ontogeny, with greater villi length and goblet cell abundance in the anterior intestine at 66 dph. In conclusion, the novel D1 feeding protocol, incorporating barnacle nauplii and a microdiet richer in vegetable phospholipids, enhanced larval quality by effectively reducing skeletal anomalies and accelerating internal organ development. Full article

Due to the rising atmospheric carbon dioxide levels driven by human activity, extensive scientific efforts have been dedicated to developing methods aimed at reducing its concentration in the atmosphere. A novel approach involves using hydrates as a long-lasting reservoir of CO₂ sequestration. This review provides an initial overview of hydrate characteristics, their formation mechanisms, and the experimental techniques commonly employed for their characterization, including X-ray, Raman spectroscopy, cryoSEM, DSC, and molecular dynamic simulation. One of the main challenges in CO₂ sequestration via hydrates is the requirement of high pressures and low temperatures to stabilize CO₂ molecules within the hydrate crystalline cavities. However, deviations from classical temperature-pressure phase diagrams observed in natural and engineered environments can be explained by considering that hydrate stability and formation are primarily governed by chemical potentials, not just temperature and pressure. Activity, which reflects concentration and non-ideal interactions, greatly influences chemical potentials, emphasizing the importance of solution composition, salinity, and additives. In this context the role of promoters and inhibitors in facilitating or hindering hydrate formation is discussed. Furthermore, the review presents an overview of the impact of marine sediments and naturally occurring compounds on CO₂ hydrate formation, along with the sampling methodologies used in sediments to determine the composition of these natural compounds. Special attention is given to the effect and chemical characterization of dissolved organic matter (DOM) in marine aquatic environments. The focus is placed on the key roles of various natural occurring molecules, such as amino acids, protein derivatives, and humic substances, along with the analytical techniques employed for their chemical characterization, highlighting their central importance in the CO₂ gas hydrates formation. Full article

AA1050 aluminum was hydrostatically extruded at room temperature to true strains of 0.9 and 3.2, and at cryogenic temperature to a true strain of 0.9. As a result of the extrusion process, the yield strength (YS) increased by 130–160% to 120–130 MPa, and the ultimate tensile strength (UTS) rose by 64–81% to 125–140 MPa. The hardness reached 46–49 HV. YS and UTS values correspond to mechanical properties typical of the H6 or H8 temper designations, with unusually high elongation at break ranging from 15% to 16.4%. Differences in lattice parameters, crystallite size, and lattice strain between samples deformed under various conditions—as well as those annealed after deformation—were within the margin of measurement uncertainty. This indicated that differences in defect density between the samples were relatively small, due to dynamic recovery occurring during extrusion. However, positron annihilation spectroscopy demonstrated that the cryo-cooled material extruded at a true strain of 0.9, as well as the one extruded at RT at a true strain of 3.2, exhibited significantly higher mean lattice defect concentrations compared to the sample extruded at RT at a true strain of 0.9. The predominant defects detected were vacancies associated with dislocations. The extrusion parameters also significantly affected the crystallographic texture. In particular, they altered the relative proportions of the <111> and <100> components in the axial texture, with the <100> component becoming dominant in cryogenically extruded samples. This trend was further intensified during recrystallization, which enhanced the <100> component even more. Recrystallization of the deformed materials occurred in the temperature range of 520–570 K. The activation energy for grain boundary migration during recrystallization was estimated to be approximately 1.5 eV. Full article

Parkinson’s disease, associated with mutations in the GBA1 gene (GBA1-PD), is the most common genetic form of Parkinson’s disease (PD), marked by clinical heterogeneity influenced by mutation type. Extracellular vesicles (EVs), key mediators of intercellular communication, are implicated in PD pathogenesis through the transport of pathological proteins and lipids. In this study, we analyzed blood plasma-derived EVs from GBA1-PD patients carrying p.N370S and p.L444P mutations and from healthy controls using cryo-electron microscopy, lipidomics, and proteomics. EVs from GBA1-PD patients were significantly larger than those from controls, with the largest size and most multilayered vesicles observed in p.N370S carriers. Lipidomic profiling identified 237 lipid species; of these, 186 lipids were altered in p.N370S and 24 in p.L444P versus controls. Mutation carriers showed distinct lipid signatures, with p.L444P samples enriched predominantly in sphingolipids, while p.N370S carriers exhibited more extensive lipid remodeling across multiple classes, including triglycerides, cholesteryl esters, and phospholipids. Notably, Cer 23:0 was elevated across all GBA1-PD groups. Proteomic analysis revealed enrichment in pathways related to lipid transport, immune regulation, and vesicle-mediated processes. Overall, GBA1-PD patients share a distinct lipidomic EV signature, with mutation-specific patterns reflecting differing mechanisms of lysosomal dysfunction. These findings support the potential of EV profiling to unravel disease heterogeneity and identify biomarkers. Full article

Detergent solubilisation remains the most commonly used but potentially problematic method to extract membrane proteins from lipid bilayers for Cryo-EM studies. Although recent advances have introduced excellent alternatives—such as amphipols, nanodiscs and SMALPs—the use of detergents is often necessary for intermediate steps. In this paper, we share our experiences working with detergent-solubilised samples within the modern Cryo-EM structural pipeline from the perspective of an EM specialist. Our aim is to inform novice users about potential challenges they may encounter. Drawing on specific examples from a variety of biological membrane systems, including Magnesium channels, lipopolysaccharide biosynthesis, and the human major facilitator superfamily transporters, we describe how the intrinsic properties of detergent-extracted samples can affect protein purification, Cryo-EM grid preparation (including the formation of vitreous ice) and the reconstitution of proteins into micelles. We also discuss how these unique characteristics can impact different stages of structural analysis and lead to complications in single-particle averaging software analysis. For each case, we present our insights into the underlying causes and suggest possible mitigations or alternative approaches. Full article

Effective control of calcium carbonate (CaCO₃) scale formation is crucial to improve the performance and economic efficiency of water systems. This study investigates the impact of various scale inhibitors on the nucleation and crystallization processes of CaCO₃. Calcium carbonate particles were synthesized by mixing CaCl₂·2H₂O and NaHCO₃ solutions, in the presence of various scale inhibitors that had not previously been investigated using the experimental techniques employed in this study. Particle size distribution and zeta potential were analyzed using dynamic light scattering (DLS), while Ca⁺² consumption and pH changes were monitored with ion-selective electrodes. Crystal morphology was evaluated using scanning electron microscopy (SEM) and cryo-transmission electron microscopy (cryo-TEM). We demonstrated that, in all samples, approximately 98% of the CaCO₃ particles (sized between 400 and 840 nm) are formed within the first 30 min of synthesis, and these particles then aggregate to form larger particles (840–1100 nm in size). Due to the solution’s high supersaturation, the inhibitors influence calcium consumption only after 5 min of synthesis. All inhibitors, especially DTPMP, decrease calcium consumption and particle size during synthesis. The zeta potential and morphology of the particles in the samples containing inhibitors differed from those in the control group. Cryo-TEM observations revealed distinct nanometric precursor phases in the calcite crystallization process without inhibitors and different nanostructures when scale inhibitors were used. Moreover, conchoidal fractures were observed in the nanoparticles formed in the presence of DTPMP. This study demonstrates the effectiveness of various inhibitors in reducing calcium consumption in solution and altering the morphology of CaCO₃ crystals, thereby preventing calcium carbonate (CaCO₃) scale formation. Full article

Proteins that span cellular membranes represent around 30% of the proteome and over 50% of drug targets. A variety of synthetic and naturally-occurring small organic molecules interact with membrane proteins and up- and down-regulate protein function. The atomic details of these regulatory molecules offer important information about protein function and aid the discovery, refinement and optimization of new drugs. X-ray crystallography and cryo-electron microscopy (cryo-EM) are not always able to resolve the structures of small molecules in their physiological sites on membrane proteins, particularly if the molecules are unstable or are reactive enzyme substrates. Solid-state nuclear magnetic resonance (SSNMR) is a valuable technique for filling in missing details on the conformations, dynamics and binding environments of small molecules regulators of membrane proteins. SSNMR does not require diffracting crystals possessing long-range order and can be performed on proteins within their native membranes and with freeze-trapping to maintain sample stability. Here, work over the last two decades is described, in which SSNMR methods have been developed to report on interactions of the ATP substrate with the Na,K-ATPase (NKA), an ion-transporting enzyme that maintains cellular potential in all animals. It is shown how a combination of SSNMR measurements on membranous NKA preparations in the frozen and fluid states have provided unique information about the molecular conformation and local environment of ATP in the high-affinity nucleotide site. A combination of chemical shift analysis using density functional theory (DFT) calculations, dipolar coupling measurements using REDOR and measurements of the rates of proton spin diffusion is appraised collectively. The work described herein highlights the methods developed and challenges encountered, which have led to a detailed and unrivalled picture of ATP in its high-affinity binding site. Full article

Extracellular vesicles (EVs) are key mediators of intercellular communication and play a vital role in cancer progression. While EVs in the blood are well-studied, those in local body fluids, such as gastric juice (GJ), remain underinvestigated. Previously, we first characterized GJ-derived EVs and demonstrated their potential for gastric cancer (GC) screening. Here, we conducted a detailed morphological analysis of GJ-EVs using cryo-electron microscopy, identifying both typical and atypical EV subtypes, and categorized their relative abundances. A subsequent comparison of the size distribution of GJ-derived EVs by nanoparticle tracking analysis revealed significant differences between samples obtained from GC patients (n = 40) and healthy subjects (n = 25). Additionally, the mean EV sizes differed significantly according to the presence of the tetraspanin protein CD9. Furthermore, the ratio of CD9-positive to CD9-negative EV samples differed between cancer patients and healthy donors. These data suggest that GJ contains distinct subpopulations of EVs that vary in size and CD9 expression, as well as EVs with certain types of atypical morphology. The identification of discrepancies in EV size and the presence of CD9 between GJ from cancer patients and healthy individuals offers potential avenues for the identification of new GC markers. Full article

The solubility of soybean isolate protein (SPI) undergoes significant degradation during storage and transportation. This study investigates the formulation and assessment of SPI-stabilized medium internal phase emulsions (MIPEs) with different solubilities, namely SPI80, SPI70, SPI60, and SPI50, corresponding to solubility levels of about 80%, 70%, 60%, and 50%, respectively. The contact angles of these SPI variants ranged from 79.35 to 86.55 degrees, with SPI60 and SPI50 exhibiting significantly higher values compared to SPI80 and SPI70. All SPI samples were successfully utilized for the preparation of MIPEs. However, as SPI solubility decreases, emulsion stability progressively declines, accompanied by a reduction in the absolute value of zeta potential. Additionally, interfacial protein adsorption in emulsions decreases with decreasing SPI solubility, a trend that is similarly observed in viscosity characteristics, storage modulus (G′), and loss modulus (G″). Confocal laser scanning microscopy (CLSM) and cryo-scanning electron microscopy (Cryo-SEM) analyses revealed that emulsions exhibit reduced uniformity and a less interconnected microstructural network as SPI solubility decreases. These findings provide a theoretical foundation for utilizing low-solubility SPI in MIPEs applications. Full article

Viral infections in stone fruit crops cause substantial economic losses across all sectors of production. Despite their significance, viruses affecting stone fruits remain under-investigated in Kazakhstan. Among these, plum pox virus (PPV, genus Potyvirus, family Potyviridae), commonly known as Sharka, is the most critical viral pathogen worldwide, severely threatening the sustainable cultivation of stone fruits and posing risks to food security. This study aimed to evaluate virus management strategies in stone fruit crops to facilitate the production of healthy planting material from valuable genotypes. Field surveys were conducted in plum and apricot orchards located in the Almaty region (Southeast Kazakhstan) and the Saryagash region (Southern Kazakhstan). Plant samples were tested for the presence of the following viruses: apple chlorotic leaf spot virus (ACLSV), apple mosaic virus (ApMV), PPV, prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), cherry green ring mottle virus (CGRMV), and myrobalan latent ringspot virus (MLRSV). Real-time RT-PCR diagnostics confirmed the presence of PPV in the ‘Stanley’ and ‘Ansar’ cultivars and Prunus armeniaca genotypes, while both PPV and ACLSV were detected in the ‘Ayana’ variety. Chemotherapy (Ribavirin), thermotherapy, cryotherapy, and shoot apical meristem (SAM) culture, both individually and in combination, were used to eliminate viruses and regenerate virus-free plants. Successful virus eradication was achieved for PPV and ACLSV. However, the ‘Stanley’ and ‘Ansar’ cultivars did not survive the treatment process, likely due to high thermo- or cryo-sensitivity. As a result of this research, an in vitro collection of virus-free plants was established, comprising eight rootstocks, six plum cultivars, and three apricot genotypes. Full article