Search Results (203)

Cryopreservation of ram semen is an essential tool in assisted reproductive technology; however, oxidative stress generated during the freezing process may compromise sperm quality. This study evaluated the effects of Se and SeNPs on post-thaw sperm quality, PRDX5 expression, and oxidative status in cryopreserved ram semen. In this study, semen samples collected from five mature rams (three collections at 2-week intervals, yielding a total of 15 ejaculates) were frozen in liquid nitrogen using extenders supplemented with selenium (1 μg/mL, S1; 10 μg/mL, S2) or selenium nanoparticles (SeNPs; 1 μg/mL, N1; 2 μg/mL, N2) alongside a nonsupplemented control extender. Post-thaw sperm quality was evaluated using computer-assisted sperm analysis (CASA) for motility, kinematic parameters, viability, membrane integrity (HOST) assays, chromatin condensation assessment, and morphological analysis. Total oxidant status (TOS) measurements and PRDX5 gene expression analysis were performed separately. Low-dose SeNPs (1 µg/mL) significantly improved total motility (55.73 ± 19.01%), progressive motility (25.05 ± 15.34%), viability (57.27 ± 19.30%), HOST-positive spermatozoa (50.87 ± 18.91%), and morphologically normal spermatozoa (88.27 ± 4.10%) compared with the control and high-dose sodium selenite groups (p < 0.05). Chromatin condensation abnormalities were lowest in the SeNP-treated group. S1 and N2 also improved motility and morphology compared with the control; however, the increases were numerically smaller than those observed in the N1 group. In contrast, S2 supplementation showed limited benefit, with values that were similar to those of the control. Morphologically normal spermatozoa were highest in N1, followed by S1 and N2, while S2 and the control exhibited the lowest values (p < 0.05). In contrast, no significant differences were detected in TOS or PRDX5 gene expression among the experimental groups (p > 0.05). These findings indicate that low-dose SeNPs enhance post-thaw sperm functional integrity and cryotolerance without inducing measurable changes in bulk oxidative markers or gene transcription. Full article

Additive manufacturing of hydrogel-based scaffolds requires concurrent control of material rheology and extrusion dynamics, especially in multi-material architectures. In this work, we develop a modular multi-material extrusion-based 3D-printing platform that combines a filament-fed extruder for thermoplastic polymers with a piston-driven extruder for viscous gel inks, together with an empirical calibration procedure for gel dosing. The calibration algorithm optimizes the pre-extrusion and retraction displacement (E_Pr/R) based on stepwise extrusion experiments and reduces the discrepancy between theoretical and measured deposited mass for shear-thinning alginate gels to below the prescribed tolerance. The calibrated system is then used to fabricate two representative hybrid constructs: partially crosslinked sodium alginate scaffolds with an internal hollow channel supported by a removable polycaprolactone framework, and self-supporting structures based on a sodium alginate–chitosan polyelectrolyte complex obtained by sequential co-extrusion. The resulting constructs remain mechanically stable after ionic crosslinking and solvent treatment and can subsequently be converted into highly porous scaffolds by freeze- or supercritical drying. The proposed combination of hardware architecture and extrusion calibration enables reproducible multi-material 3D printing of hydrogel–thermoplastic hybrid scaffolds and can be readily adapted to other gel-based inks for tissue engineering applications. Full article

Semen cryopreservation is a critical biotechnological approach for preserving superior genetic resources in livestock. Spermatozoa are particularly vulnerable to cryogenic stress during the freeze–thaw process, resulting in impaired structure and function. Therefore, the development of effective cryoprotective additives is essential for improving yak semen cryopreservation. In this study, ginseng peptide (GFREH) was incorporated into the freezing extender at different concentrations (0, 0.25, 0.5, 0.75, and 1.0 mg/mL) to evaluate its effects on post-thaw sperm quality, in vitro fertilization (IVF) capacity, and the underlying regulatory mechanisms. Semen samples treated with 0 and 0.75 mg/mL GFREH were further subjected to proteomic analysis to elucidate the molecular basis of its cryoprotective action. The results demonstrated that GFREH significantly increased total motility (TM), progressive motility (PM), straight-line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), as well as plasma membrane and acrosome integrity of frozen–thawed yak spermatozoa (p < 0.05). GFREH also significantly reduced malondialdehyde (MDA) levels while enhancing antioxidant enzyme activities, mitochondrial membrane potential (MMP), and ATP content (p < 0.05). Moreover, GFREH at concentrations of 0.5, 0.75, and 1.0 mg/mL significantly improved IVF and blastocyst formation rates compared with the control (p < 0.05), with the 0.75 mg/mL group exhibiting the highest fertilization and blastocyst rates. Proteomic analysis further revealed that GFREH modulated the PI3K/AKT signaling pathway and downregulated FOXO1 expression. Collectively, these findings indicate that ginseng peptides enhance yak sperm cryotolerance by coordinating oxidative balance, mitochondrial energy metabolism, and survival-related signaling, with 0.75 mg/mL representing an optimal effective concentration within the functional dose range tested. Full article

Background: Self-amplifying mRNA (sa-mRNA) represents a promising platform for vaccines and gene therapies, offering sustained protein expression at low doses through self-replication. For vaccines targeting respiratory pathogens, pulmonary delivery of sa-mRNA lipid nanoparticles (LNPs) is particularly advantageous, enabling direct delivery to the infection site and induction of mucosal immunity. Objective: In this study, we evaluated the stability of sa-mRNA–LNPs under refrigerated and frozen conditions and developed a dry powder formulation suitable for inhalation, produced by freeze-drying followed by cryomilling with leucine. Methods: sa-mRNA–LNPs formulated in HEPES buffer with 20% (w/v) sucrose were stored for up to 8 weeks as liquid or freeze-dried samples at various temperatures (−80 °C, −20 °C, 4 °C, and 20 °C). Biological stability was assessed by transfection efficiency in HeLa cells, while physical stability was characterized by encapsulation efficiency, zeta potential, particle size, and polydispersity index. Results: Liquid formulations remained stable for at least 8 weeks at −80 °C and −20 °C but rapidly lost stability at 4 °C and 20 °C. Freeze-drying effectively preserved sa-mRNA–LNP functionality and structural integrity for up to 8 weeks at 4 °C, with only minor structural changes. Subsequent cryomilling in the presence of 4 wt-% leucine produced a respirable dry powder while retaining approximately 60% of the original sa-mRNA–LNP functionality. Although cryomilling induced some structural alterations, the remaining functional fraction remained stable during storage. The resulting powders displayed favorable aerosol performance for deep lung delivery, as demonstrated by cascade impaction (MMAD = 4.13 ± 0.26 µm). Conclusions: In conclusion, freeze-drying effectively preserved sa-mRNA–LNP integrity at 4 °C, whereas cryomilling with leucine produced a respirable dry powder suitable for pulmonary delivery, providing a foundation for globally accessible, needle-free sa-mRNA vaccines against respiratory diseases. Full article

This study evaluated the effects of varying dietary salt and boric acid addition doses on the milk quality of Savak Akkaraman sheep. A total of 120 animals were as-signed to six treatment groups (n = 20): control (C), rock salt (S; 10 g/day), boric acid 20 mg/day (B20), boric acid 40 mg/day (B40), BS20 (20 mg boric acid + 10 g/day rock salt), and BS40 (40 mg boric acid + 10 g/day rock salt). All analyses were performed in duplicate on six samples, taken on days 30 and 35 following the administration of the additives. Physicochemical analyses only showed significant variation in milk pH (p = 0.006), while acidity, dry matter, and ash remained unaffected. Strong positive correlations were found among protein, lactose, salt, and density (r > 0.95; p < 0.001). Coagulation times differed widely, with the longest being observed in BS20 (995.03 s) and the shortest in BS40 (141.73 s). Among mineral parameters, only selenium levels differed significantly between the treatment groups (p < 0.05). No significant differences were found for fat, solids-not-fat, lactose, freezing point, or electrical conductivity. Importantly, boron addition had a significant influence on total casein content (p < 0.001). Overall, dietary rock salt and boric acid did not markedly alter the basic milk composition but produced notable physicochemical changes, particularly in coagulation behavior and casein levels, which may influence the technological properties of sheep milk. Full article

Background/Objectives: Therapeutic drug monitoring (TDM) of β-lactams (BL), BL/β-lactamase inhibitor (BLI) combinations (BL/BLIc), and of fosfomycin may play a key role in optimizing antimicrobial therapy and in preventing resistance development, especially when used by continuous infusion in critically ill or immunocompromised patients. Unfortunately, analytical methods for simultaneously quantifying multiple BL/BLIc in plasma are still lacking. Methods: The aim of this study was to develop and validate two rapid, sensitive, and accurate UPLC–qTOF–MS/MS methods for the simultaneous quantification of five novel β-lactam or β-lactam/β-lactamase inhibitor combinations (ceftolozane/tazobactam, ceftazidime/avibactam, meropenem/vaborbactam, cefiderocol, and ceftobiprole) along with fosfomycin. Methods: Human plasma samples were prepared by protein precipitation using methanol containing isotopically labeled internal standards. Chromatographic separation was achieved within 10–12 min using two Agilent Poroshell columns (EC-C18 and PFP) under positive and negative electrospray ionization modes. The method was validated according to the EMA guidelines by assessing selectivity, linearity, precision, accuracy, matrix effects, extraction recovery, and stability. Results: The methods exhibited excellent linearity (R² ≥ 0.998) across the calibration ranges for all of the analytes (1.56–500 µg/mL), with limits of quantification ranging from 1.56 to 15.62 µg/mL. Intra- and inter-day precision and accuracy were always within ±15%. Extraction recovery always exceeded 92%, and the matrix effects were effectively corrected through isotopic internal standards. No carry-over or isobaric interferences were observed. All the analytes were stable for up to five days at 4 °C, but the BL and BL/BLIc stability was affected by multiple freeze–thaw cycles. Conclusions: These UPLC-qTOF-MS/MS multi-analyte methods enabled a simultaneous, reliable quantification in plasma of five novel beta-lactams and of fosfomycin. Robustness, high throughput, and sensitivity make these multi-methods feasible for real-time TDM, supporting personalized antimicrobial dosing and improved therapeutic outcomes in patients with severe or multidrug-resistant infections. Full article

Valorization of black carrot pomace (BCP), an industrial by-product rich in bioactive compounds, was performed using sustainable extraction and formulation approaches. Bioactive compounds were extracted, using water as a solvent, via ultrasonic processing. The resulting liquid extract (BCP-E) was then freeze-dried with a gum Arabic gel system to obtain a powder formulation (FD-BCP). The technological, physicochemical, and bioactive characteristics of both formulations are described. Total monomeric anthocyanin and antioxidant activities (DPPH and ABTS) did not differ substantially (p > 0.05), but the liquid extract’s total phenolic content was significantly higher (4.95 mg GAE/g db) than the powder formulation’s (4.46 mg GAE/g db). While FD-BCP had three main hydrophilic phenolic compounds, suggesting partial encapsulation, high-resolution LC-MS analysis identified 21 phenolic compounds in BCP-E, dominated by chlorogenic, quinic, and protocatechuic acids. The development of a stable gum Arabic matrix that maintains the phenolics’ structural integrity was confirmed by SEM and FTIR observations. According to cytotoxicity tests conducted on L929 fibroblast cells, both formulations were biocompatible (>70% viability) and even stimulated cell growth at moderate dosages. Dose- and time-dependent viability patterns were successfully described by Principal Component Analysis and Artificial Neural Network models, highlighting the fact that formulation type is the main factor influencing biological response. Overall, ultrasonic extraction and freeze-drying offer efficient and sustainable strategies for producing stable and bioactive-rich components from black carrot pomace that may be used in functional foods and biomedical products. Full article

To address the engineering challenge of durability deterioration in concrete structures in the cold and saline regions in northern China, this study investigated PVA fiber-reinforced concrete under combined sulfate attack and freeze–thaw cycles using PVA fiber volume fractions (0%, 0.1%, 0.3%, 0.5%) and salt-freeze cycles (0, 25, 50, 75, 100, 125, 150 cycles) as key variables. By testing the mechanical and microscopic properties of the specimens after salt-freeze, the degradation law of macroscopic performance and the evolution mechanism of microscopic structure of PVA fiber concrete under different volume fractions are analyzed, and the salt-freeze damage evolution equation is established based on the loss rate of relative dynamic elastic modulus. The results show that the addition of PVA fibers has no significant inhibitory effect on the surface erosion of concrete, and the degree of surface spalling of concrete still increases with the increase in the number of salt-freeze cycles. With the increase in the number of salt-freezing cycles, the mass, relative dynamic elastic modulus and cube compressive strength of the specimens first increase and then decrease, while the splitting tensile strength continuously decreases. The volume fraction of 0.3% PVA fibers has the most significant effect on improving the cube compressive strength and splitting tensile strength of concrete, and at the same time, it allows concrete to reach its best salt-freezing resistance. PVA fibers contribute to a denser microstructure, inhibit the development of micro-cracks, delay the formation of erosion products, and enhance the salt-freezing resistance of concrete. The damage degree D of relative dynamic elastic modulus for PVA fiber concrete exhibits a cubic functional relationship with the number of salt-freeze cycles N, and the correlation coefficient R² is greater than 0.88. The equation can accurately describe the damage and deterioration law of PVA fiber concrete in the salt-freeze coupling environment. In contrast to numerous studies on single-factor exposures, this work provides new insights into the degradation mechanisms and optimal fiber dose for PVA fiber concrete under the synergistic effect of combined sulfate and freeze-thaw attacks, a critical scenario for infrastructure in cold saline regions. This study can provide theoretical guidance for the durability assessment and engineering application of PVA fiber concrete in cold and saline regions. Full article

Seaweeds are emerging renewable biomass resources rich in valuable phytochemicals; however, effective stabilization strategies are required to enable their incorporation into sustainable food and bioprocessing applications. This study investigated the effects of convective drying (40–80 °C) and freeze-drying on the chemical composition and functional properties of the underexplored red seaweed Sarcodiotheca gaudichaudii. The drying method significantly modulated nutrient retention, pigment stability, and bioactivity. Freeze-drying and high-temperature convective drying (≥70 °C) resulted in higher protein and saturated fatty acid contents but led to substantial losses of pigments and antioxidant capacity. In contrast, moderate convective drying (40–60 °C) favored the retention of minerals, polyunsaturated fatty acids, essential amino acids, and pigments, while enhancing total phenolic and flavonoid contents and improving antioxidant performance (DPPH and ORAC). All extracts exhibited dose-dependent α-glucosidase inhibition (25–58%) within a concentration range of 0.10–40.0 mg/mL, with freeze-dried samples showing the strongest inhibitory effect. Similarly, cytotoxicity assays conducted on A549 and AGS cancer cell lines at concentrations of 1.25–40.0 mg/mL revealed that freeze-dried extracts consistently displayed the lowest IC₅₀ values. Overall, convective drying better preserved nutritional quality, whereas freeze-drying maintained higher biological functionality, revealing a process-dependent trade-off relevant to industrial biomass stabilization and functional ingredient development. Full article

Evidence increasingly indicates that synaptic vesicle dysfunction emerges early in Parkinson’s disease (PD), preceding overt dopaminergic neuron loss rather than arising solely as a downstream consequence of neurodegeneration. α-Synuclein (αSyn), a presynaptic protein that regulates vesicle clustering, trafficking, and neurotransmitter release under physiological conditions, exhibits dose-, conformation-, and context-dependent actions that distinguish its normal regulatory roles from pathological effects observed in disease models. This narrative review synthesizes findings from a structured search of PubMed and Scopus, with emphasis on α-syn-knockout (αSynKO) and BAC transgenic (αSynBAC) mouse models, which do not recapitulate the full human PD trajectory but provide complementary insights into αSyn physiological function and dosage-sensitive vulnerability. Priority was given to studies integrating ultrastructural approaches—such as cryo-electron tomography, high-pressure freezing/freeze-substitution TEM, and super-resolution microscopy—with proteomic and lipidomic analyses. Across these methodologies, several convergent presynaptic alterations are consistently observed. In vivo and ex vivo studies associate αSyn perturbation with impaired vesicle acidification, consistent with altered expression or composition of vacuolar-type H⁺-ATPase subunits. Lipidomic analyses reveal age- and genotype-dependent remodeling of vesicle membrane lipids, particularly curvature- and charge-sensitive phospholipids, which may destabilize αSyn–membrane interactions. Complementary biochemical and cell-based studies support disruption of SNARE complex assembly and nanoscale release-site organization, while ultrastructural analyses demonstrate reduced vesicle docking, altered active zone geometry, and vesicle pool disorganization, collectively indicating compromised presynaptic efficiency. These findings support a synapse-centered framework in which presynaptic dysfunction represents an early and mechanistically relevant feature of PD. Rather than advocating αSyn elimination, emerging therapeutic concepts emphasize preservation of physiological vesicle function—through modulation of vesicle acidification, SNARE interactions, or membrane lipid homeostasis. Although such strategies remain exploratory, they identify the presynaptic terminal as a potential window for early intervention aimed at maintaining synaptic resilience and delaying functional decline in PD. Full article

Game meats are particularly prone to oxidation and microbial spoilage due to their specific characteristics and the procedures required to obtain them. Various sustainable bioactive molecules derived from food industry by-products, such as olive mill wastewater, have the potential to enhance the stability and safety of game meats. The use of different levels of polyphenolic extracts from olive mill vegetation water, encapsulated through a freeze-drying process, was tested on wild boar meat patties as an antioxidant and antimicrobial. Two separate trials were performed. Trial 1 was carried out by adding different concentrations of polyphenolic extract (0, 1, and 2%) during the production of wild boar patties, and trial 2 by adding 1.5% salt and adding or not adding 2% polyphenolic extract. The first trial revealed antioxidant effects on the raw patties during storage time, both on colour (increasing in saturation index) and thiobarbituric acid-reactive substances (0.306, 0.268, and 0.254 mg MDA/kg after 5 days of storage in the control with 1% and 2% polyphenolic extract groups, respectively). Oxidation was also reduced during cold storage of cooked patties. Trial 1 also revealed a dose-dependent antimicrobial effect, mainly on Enterobacteriaceae and Pseudomonas spp. Trial 2 confirmed that salt plus extract addition had an overall higher antimicrobial effect than when singularly added, but with a moderate increase in the hardness of the products. Full article

Lung cancer remains one of the leading causes of cancer-related mortality worldwide, with therapeutic efficacy often hindered by the development of multidrug resistance. Consequently, alternative strategies to slow down tumor progression warrant rigorous investigation. Bioactive molecules derived from tissues and organs have shown potential therapeutic properties for several diseases. We investigated the biological role of soluble bioactive factors derived from lyophilized porcine freeze-dried lung tissue (FDLT), as they may contain tumor-suppressing components involved in the progression of non-small cell lung cancer (NSCLC). NSCLC H1975 and PC9 cell lines were treated with FDLT at concentrations of 0.25 mg/mL and 0.5 mg/mL. Cell cycle analysis and mitochondrial membrane potential (MMP) assays were performed to assess cell proliferation and cell death activation. In parallel, epithelial–mesenchymal transition (EMT) markers were detected by qRT-PCR. Our findings showed that FDLT treatment reduced the viability of H1975 and PC9 cells in a dose-dependent manner, along with significant suppression of cell proliferation and colony formation. Moreover, FDLT treatment altered the cell cycle phases and determined a concomitant reduction of cyclin D1 levels as well as induction of mitochondria depolarization by suppressing MMP. Finally, qRT-PCR revealed significant downregulation of EMT-related genes vimentin and N-cadherin, along with the EMT transcription factor Twist. These findings highlight soluble FDLT-derived biomolecules as a potential tool to design alternative treatment strategies for NSCLC. Full article

Background/Objectives: Natural plant-based compounds, especially black pepper extract, are known to have anti-inflammatory, antibacterial, and antioxidant qualities that promote procollagen formation and wound healing. This study focused on developing a collagen-based composite enriched with P. nigrum extract in powder form, designed to enhance the efficacy of the antibiotic cefazolin while promoting the healing of chronic wounds. Methods: The polyphenolic P. nigrum extract was obtained by ultrasound-assisted extraction and was characterised by UHPLC-MS/MS and spectrophotometry. Antimicrobial and antioxidant activities were assessed using conventional methods. Pharmacokinetic and pharmacodynamic parameters were evaluated for the specific taxon compounds using Deep-RK. P. nigrum extract was incorporated into a collagen hydrogel with arginine and freeze-dried. The powders were characterised by FTIR, SEM, TGA-DSC, and DLS. The antimicrobial activity and potential synergistic effects with cefazolin were evaluated on reference microbial strains and isolates from infected wounds. Biocompatibility and hemocompatibility were evaluated, as well as wound closure in vitro. Results: Polyphenols, including phenolic acids, stilbenes, anthocyanins, and flavonoids, which provide a potent antioxidant capacity through electron transfer mechanisms (FRAP, CUPRAC), were abundant in the P. nigrum extract. FTIR and SEM analyses confirmed the integration of phenolic compounds into the collagen–arginine matrix without protein denaturation. TGA–DSC data showed thermal stabilisation at moderate extract concentrations. The extract exhibited predominantly bacteriostatic antibacterial activity and antibiofilm effects, with synergy/additivity with cefazolin, especially at medium doses. Tests on keratinocytes confirmed biocompatibility, and hemocompatibility demonstrated an excellent safety profile, with protection against AAPH-induced oxidative stress. Conclusions: Overall, collagen powders with P. nigrum extract at moderate/low concentrations combine stability, antibiotic-enhanced activity, and cellular compatibility, making them promising adjuvants for the topical treatment of chronically infected wounds. Full article

Sperm cryopreservation is a key technique in assisted reproductive technologies (ART), livestock breeding, fertility preservation, and wildlife conservation. However, the freeze–thaw process induces significant oxidative stress through the production of reactive oxygen species (ROS) by mitochondria, which can lead to impaired sperm motility, membrane damage, DNA fragmentation, and reduced fertilization potential. MitoQ is a mitochondria-targeted antioxidant consisting of a ubiquinone moiety conjugated to triphenylphosphonium (TPP⁺). MitoQ selectively accumulates in the mitochondrial matrix, where it efficiently scavenges reactive oxygen species (ROS) at their point of origin. This targeted action helps preserve mitochondrial function, sustain ATP production, and inhibit apoptotic signaling. Extensive experimental evidence across diverse species, including bulls, rams, boars, humans, dogs, and goats, shows that MitoQ supplementation during cryopreservation enhances post-thaw sperm viability, motility, membrane integrity, and DNA stability. Optimal dosing between 50 and 150 nM achieves these benefits without cytotoxicity, although higher doses may paradoxically increase oxidative damage. Compared to conventional antioxidants, MitoQ offers superior mitochondrial protection and enhanced preservation of sperm bioenergetics. Future directions involve exploring synergistic combinations with other cryoprotectants, advanced delivery systems such as nanoparticles and hydrogels, and detailed mechanistic studies on long-term effects. Overall, MitoQ represents a promising adjunct for improving sperm cryopreservation outcomes across clinical, agricultural, and conservation settings. Full article

While methane emissions from cattle contribute to greenhouse gases, supplementing with red seaweed Asparagopsis taxiformis (AT) demonstrates an up to 90% methane reduction in controlled feeding studies. However, methods for delivery of AT in grazing systems remain unexplored. This study evaluated AT with mineral supplementation to 112 weaned steers grazing on annual rangeland over 157 days. Cattle were randomly assigned to access mineral with freeze-dried AT (targeting 150 mg bromoform/head/day) or mineral without AT. Methane emissions were measured using laser methane detection (LMD) and body weight, mineral consumption, and blood selenium levels were monitored. Average daily mineral consumption was lower than targeted, resulting in suboptimal bromoform intake (89.2 mg/head/day). No significant differences were observed between treatments for mineral consumption, weight gain, or blood selenium levels. Cattle with access to mineral with AT had lower peak emissions than control cattle when measured at day 25, but no differences in peak emissions were measured at day 115 or day 157. The lack of methane reduction was attributed to insufficient bromoform dosing, potential compound degradationduring field storage, and limitations of laser methane detection. Achieving consistent dosing and accurate methane assessment in extensive grazing systems requires improved delivery mechanisms, compound stabilization, and measurement techniques. Full article