Search Results (350)

Oxidized phospholipids (OxPLs) are increasingly recognized as biologically active lipids involved in various pathologies. Both exposure to pathogenic factors and the efficacy of protective mechanisms are critical to disease development. In this study, we characterized an immunoassay that quantified the total capacity of the plasma to degrade or mask OxPLs, thereby preventing their interaction with cells and soluble proteins. OxLDL-coated plates were first incubated with human blood plasma or a control vehicle, followed by an ELISA using a monoclonal antibody specific to oxidized phosphatidylethanolamine. Pretreatment with the diluted blood plasma markedly inhibited mAb binding. The masking assay was optimized by evaluating the buffer composition, the compatibility with various anticoagulants, potential interfering compounds, the kinetic parameters, pre-analytical stability, statistical robustness, and intra- and inter-individual variability. We propose that this masking assay provides a simple immunological approach to assessing protective mechanisms against lipid peroxidation products. Establishing this robust and reproducible method is essential for conducting clinical association studies that explore masking activity as a potential biomarker of the predisposition to a broad range of lipid-peroxidation-related diseases. Full article

Ruminant livestock contribute significantly to methane emissions, necessitating sustainable mitigation strategies. Essential oils (EOs) show promise for modulating ruminal fermentation, but their synergistic effects remain underexplored. Two 24 h in vitro experiments evaluated the synergistic effects of EO blends on rumen microbial fermentation. Exp. 1 screened five oils using two triad combinations. Triad 1 tested 10 combinations of thyme (THY), peppermint (PPM), and cinnamon leaf (CIN) oils. Triad 2 tested 10 combinations of anise (ANI), clove leaf (CLO), and peppermint (PPM) oils. Each blend was tested at 400 mg/L, using batch culture methods measuring: pH, ammonia-N (NH₃-N), and volatile fatty acids (VFAs). The two most effective blends, designated as T1 and T2, were selected for Exp. 2 to assess total gas and methane (CH₄) production using pressure transducer methods. All treatments were incubated in a rumen fluid–buffer mix with a 50:50 forage-to-concentrate substrate (pH 6.6). In Exp. 1, data were analyzed according to the Simplex Centroid Design using R-Studio. In Exp. 2, an analysis was conducted using the MIXED procedure in SAS. Mean comparisons were assessed through Tukey’s test. The results from Exp. 1 identified CIN+PPM (80:20) and ANI+CLO (80:20) as optimal combinations, both increasing total VFAs while reducing acetate/propionate ratios and NH₃-N concentrations. In Exp. 2, both combinations significantly reduced total gas and CH₄ productions compared to the control, with CIN+PPM achieving the greatest methane reduction (similar to monensin, the positive control). Specific essential oil combinations demonstrated synergistic effects in modulating rumen fermentation and reducing methane emissions, offering potential for sustainable livestock production. Further in vivo validation is required to optimize dosing and assess long-term effects on animal performance. Full article

The mechanical properties of the extracellular matrix critically influence cell behavior in both physiological and pathophysiological states, including cardiac fibrosis. In vitro models have played a critical role in assessing biological mechanisms. In this study, we characterized mechanically tunable enzymatically crosslinked gelatin-microbial transglutaminase (mTG) hydrogels for modeling cardiovascular diseases. Gelatin hydrogels were fabricated via direct mixing or immersion crosslinking methods. Hydrogel formulations were assessed using the Piuma nanoindenter and Instron systems. This study investigates the effects of fabrication methods, UV ozone (UVO) sterilization, crosslinking methods, and incubation media on hydrogel stiffness. Further, this study examined the response of murine cardiac fibroblasts to hydrogel stiffness. The hydrogels exhibited modulus ranges relevant to both healthy and fibrotic cardiac tissues. UVO exposure led to slight decreases in hydrogel modulus, while the fabrication method had a significant impact on the modulus. Hydrogels incubated in phosphate buffered saline (PBS) were stiffer than those incubated in Medium 199 (M199), which correlated with lower pH in PBS. Fibroblasts cultured on stiffer hydrogels display enhanced smooth muscle actin (SMA) expression, suggesting sensitivity to material stiffness. These findings highlight how fabrication parameters influence the modulus of gelatin-mTG hydrogels for cardiac tissue models. Full article

(1) Background: Hemorrhagic fever with renal syndrome (HFRS) remains a prevalent zoonosis in Eurasia. Orthohantavirus puumalaense (PUUV), carried by bank voles (Myodes glareolus), is the principal zoonotic pathogen of HFRS in this region. Despite ongoing efforts to develop effective drugs and vaccines against PUUV, this challenge remains. (2) Aim: In this study, we aimed to express a large quantity of the PUUV recombinant N (rN) protein using E. coli. We also sought to develop a protocol for extracting the rN protein from pellets, solubilizing, and refolding it to restore its native form. This protocol is crucial for producing a large quantity of rN protein to develop vaccines and diagnostic tools for HFRS. (3) Methods; PUUV S segment open reading frame (ORF) coding for N protein was synthesized and cloned into the plasmid vector pET-28 (A+). The ORF was transformed, expressed and induced in BL21(DE3) pLysS E. coli strain. Subsequently, rN protein was purified using immobilized metal affinity and ion chromatography. Immune reactivity of rN protein was tested by employing in house and commercial VektoHanta-IgG kit ELISA methods (both in vitro and in vivo). (4) Results: The best conditions for scaling up the expression of the PUUV rN protein were an incubation temperature of 20 °C during a 20 h incubation period, followed by induction with 0.5 mM IPTG. The most significant protein yield was achieved when the pellets were incubated in denaturing buffer with 8M urea. The highest yield of refolded proteins was attained using non-denaturing buffer (50 mM Tris-HCl) supplemented with arginine. A final 50 μL of PUUV rN protein solution with a concentration of 7 mg/mL was recovered from 1 L of culture. The rN protein elicited an antibody response in vivo and reacted with serum taken from patients with HFRS by ELISA in vitro. (5) Conclusion: Therefore, the orthohantavirus N protein’s ability to elicit immune response in vivo suggests that it can be used to develop vaccines against PUUV after conducting in vitro and in vivo studies to ascertain neutralising antibodies. Full article

The aim of this study was to develop and characterize UV-crosslinked hydrogel matrices based on polyethylene glycol diacrylate (PEGDA), gum arabic, betaine, and sodium alginate for potential bioanalytical applications. Various physicochemical analyses were performed, including pre-polymerization emulsion stability (Multiscan), FT-IR spectroscopy, swelling behavior in physiological buffers, pH monitoring, contact angle measurements, and morphological assessment via SEM and optical microscopy. The results demonstrated that both alginate content and UV exposure time significantly influence the structural and functional properties of the hydrogels. The highest swelling ratio (2.32 g/g) was observed for the formulation containing 5% sodium alginate polymerized for 5 min (5SA_5), though this sample showed mechanical fragmentation during incubation. In contrast, the most balanced performance was achieved for the 10SA_15 formulation, which maintained structural integrity and exhibited a swelling ratio of 1.92 g/g after 9 days. The contact angle analysis revealed a surface hydrophilicity range from 50° to 100°, with the lowest angle (50°) recorded for 10SA_5, indicating high surface wettability. These findings confirm the suitability of such hydrogels for biomedical applications, particularly as absorbent, stable platforms for drug delivery or wound healing. Full article

Background: Topical statin therapy holds promise for ocular diseases, such as age-related macular degeneration, but the effective delivery to the posterior segment is limited by poor aqueous solubility, chemical instability, and ocular barriers. Cyclodextrins (CDs) can enhance statin solubility and stability; however, the behavior of CD–statin complexes in aqueous eye drops—particularly their influence on the equilibrium between the inactive lactone (ring closed) and active hydroxyacid forms (ring open)—remains unclear. This study aimed to (i) investigate how 5% and 10% (w/v) concentrations of selected CDs affect the lactone/acid equilibrium of simvastatin and atorvastatin and (ii) define formulation parameters (statin form, CD type and concentration, and pH range) for stable eye drop development. Methods: Simvastatin or atorvastatin was added to buffered solutions (pH 2.0 to pH 9.5) of RMβCD, HPβCD, γ-CD, or SBEβCD at 0%, 5%, and 10% (w/v), incubated at 23 ± 1 °C, and sampled over time for UPLC quantification of lactone and hydroxyacid forms, and rate constants for the forward and reverse reaction were calculated. Phase solubility studies were also conducted to further characterize equilibrium behavior in aqueous CD systems. Results: The lactone form was most stable at a pH of 4.5, while the hydroxyacid form prevailed at a pH ≥ 7. γ-CD and HPβCD accelerated lactone hydrolysis for both statins, whereas RMβCD exerted a stabilizing effect. Increasing the CD concentration from 5% to 10% provided minimal additional stabilization. Conclusions: These findings highlight that the precise control of the pH, an appropriate cyclodextrin choice, and the selection of the statin form are critical to developing chemically stable eye drops. Full article

Rare-earth nanoparticles (RE-NPs), particularly NaYF₄:Yb³⁺,Er³⁺, have emerged as a promising class of photoluminescent probes for bioimaging and sensing applications. These nanomaterials are characterized by their ability to absorb low-energy photons and emit higher-energy photons through an upconversion luminescence process. This process can be triggered by continuous-wave (CW) light excitation, providing a unique optical feature that is not exhibited by native biomolecules. However, the application of upconversion nanoparticles (UCNPs) in bioimaging requires systematic optimization to maximize the signal and ensure biological compatibility. In this work, we synthesized hexagonal-phase UCNPs (average diameter: 29 ± 3 nm) coated with polyacrylic acid (PAA) and established the optimal conditions for imaging human erythrocytes. The best results were obtained after a 4-h incubation in 100 mM HEPES buffer, using a nanoparticle concentration of 0.01 mg/mL and a laser current intensity of 250–300 mA. Under these conditions, the UCNPs exhibited minimal cytotoxicity and were found to predominantly localize at the erythrocyte membrane periphery, indicating surface adsorption rather than internalization. Additionally, a machine learning model (Random Forest) was implemented that classified the photoluminescent signal with 80% accuracy and 83% precision, with the signal intensity identified as the most relevant feature. This study establishes a quantitative and validated protocol that balances signal strength with cell integrity, enabling robust and automated image analysis. Full article

The COVID-19 pandemic highlighted the need for rapid, cost-effective tools to monitor transmission and immune response. We developed two novel paper-based colorimetric biosensors using glutaraldehyde as a protein dye—its first use in this context. Glutaraldehyde reacts with amino groups to generate a brown color, enabling detection of SARS-CoV-2 antibodies. Wathman filter paper was functionalized with (3-aminopropyl)triethoxysilane (APTES) to immobilize virus-like particles (VLPs) and nucleocapsid protein (N-protein) as biorecognition elements. Upon incubation with antibody-containing samples, glutaraldehyde enabled colorimetric detection using RGB analysis in ImageJ software. Both sensors showed a linear correlation between antibody concentration and RGB values in buffer and serum. The VLP sensor responded linearly within the range of 1.0–20 µg/mL (green coordinate) in 500-fold diluted serum and the N-protein sensor from 1.0–40 µg/mL (blue coordinate) in 250-fold diluted serum. Both sensors demonstrated good selectivity, with glucose causing up to 18% interference. These biosensors represent a paradigm shift, as they provide a sensitive, user-friendly, and cost-effective option for semi-quantitative serological analysis. Furthermore, their versatility goes beyond the detection of SARS-CoV-2 antibodies and suggests broader applicability for various molecular targets. Full article

Nutrients function as essential biological substrates for coastal phytoplankton growth and serve as pivotal indicators in marine environmental monitoring. The intensification of land-based nutrient sources inputs has exacerbated eutrophication in Chinese coastal water, while mechanistic understanding of differential self-purification processes among distinct land-based source nutrients (river source, domestic source, aquaculture source, and industrial source) remains limited, constraining accurate assessment of bay’s self-purification capacity. This study conducted incubation experiments in Tieshan Bay (TSB) during Summer (June 2023) and winter (January 2024), systematically analyzing the self-purification process of nutrients and associated environmental drivers. Distinct source-specific patterns emerged: river inputs exhibited maximal dissolved inorganic nitrogen (DIN) 1.390 ± 0.74 mg/L, whereas industrial discharges showed peak dissolved inorganic phosphorus (DIP) 4.88 ± 1.45 mg/L. Chlorophyll a (Chl-a) concentrations varied markedly across sources, ranging from 34.97 ± 23.37 μg/L (domestic source) to 86.63 ± 77.08 μg/L (river source). First-order kinetics demonstrated significant source differentiation (p < 0.05). River-derived DIN exhibited the highest attenuation coefficient (−0.3244 ± 0.17 d⁻¹), contrasting with industrial-sourced DIP showing maximum depletion (−0.4332 ± 0.20 d⁻¹). Correlation analysis indicated that summer was significantly associated with the impacts of three key control factors pH, dissolved oxygen, and turbidity on nutrient dynamics (p < 0.05), whereas winter exhibited a stronger dependence on salinity. These parameters collectively may modulate microbial degradation pathways and particulate matter adsorption capacities. These findings establish quantitative thresholds for coastal nutrient buffering mechanisms, highlighting the necessity for source-specific eutrophication mitigation frameworks. The differential self-purification efficiencies underscore the importance of calibrating pollution control strategies according to both anthropogenic discharge characteristics and regional hydrochemical resilience, which is of key importance for ensuring the traceability and control of land-based sources of pollution into the sea and the scientific utilization of the self-purification capacity of the bay water body. Full article

Maize (Zea mays L.) silage in the irrigated and flat areas of Italy represents the most important large ruminant feed crop due to the high dry matter yield and nutritive value per hectare. The aim of the investigation was to evaluate the chemical composition and the in vitro fermentation patterns of five maize varieties (Tiesto, R700 1, MAS 78.T, DKC 7074 and KWS Kantico) freshly chopped and preserved via ensiling. The results indicated that the chemical composition was not significantly different among varieties. The substrates were incubated for 72 h with buffered rumen fluid collected from cow. The ensiling process slightly reduced gas production and fermentation kinetics, likely due to the consumption of soluble sugars during fermentation. Organic matter loss (OM loss) differed significantly (p < 0.01) among varieties in ensiled maize, correlating with their neutral detergent fiber (NDF) content. While total volatile fatty acid (VFA) production showed no significant differences between varieties, the buffer capacity ratio (BCR), an indicator of protein degradation, varied significantly. Ammonia production (NH₃) was significantly higher in ensiled samples, supporting previous findings that ensiling increases non-protein nitrogen (NPN) due to microbial proteolysis and plant enzyme activity. The gas production profiles and fermentation rates over time showed minor differences between fresh and ensiled samples, with fresh material exhibiting faster fermentation kinetics due to the presence of soluble sugars. These findings highlight the importance of evaluating maize silage quality to optimize ruminant nutrition and feed efficiency. Full article

Growing evidence supports the use of nitrate-reducing bacterial strains as probiotics to enhance the benefits of nitrate metabolism for both oral and systemic health. This study aimed to test the nitrate reductase activity of Levilactobacillus brevis CD2 (DSM-27961/CNCM I-5566), a strain widely used as a starter culture in fermented foods and recognized for its multifaceted health-promoting probiotic properties. We also sought to determine whether the probiotic lysate enhances nitrate reduction ex vivo using six salivary samples from healthy subjects while evaluating its potential influence on pH and buffering capacity. Considering the established link between lactate metabolism and nitrite production, we assessed the salivary levels of D-lactate after a 3-hour incubation with or without Lv. brevis. The results indicate that Lv. brevis CD2 exhibits significant intrinsic and concentration-dependent nitrate reductase activity. Additionally, treatment with Lv. brevis for 3 h significantly increased nitrite generation across all saliva samples, with further enhancement observed after the addition of exogenous nitrates. Lv. brevis also significantly improved salivary pH and buffering capacity, particularly when combined with nitrate. Furthermore, the probiotic treatment resulted in reduced levels of salivary D-lactate. To further support and validate our in vitro and ex vivo findings, we evaluated the oral nitrate-reducing activity in saliva samples from healthy individuals treated for four weeks with Lv. brevis CD2 lozenges. Of note, the results indicated that the probiotic group showed a significant increase in oral nitrate-reducing capacity compared to baseline and placebo after four weeks of treatment. Overall, our study suggests that Lv. brevis CD2 acts as a nitrate-reducing probiotic, providing new insights into its health benefits and complementing findings from previous studies. Full article

Amylases facilitate the hydrolysis of starch into simpler sugars, thus playing a significant role in various industrial applications. This study aimed to isolate and characterize bacteria capable of producing amylase from soil samples collected from the Guassa Community Conservation Area (GCCA), Ethiopia. Comprehensive biochemical and morphological characterizations were performed on strains isolated from GCCA soil, followed by the optimization of amylase activity. Among the isolates, Kocuria rosea and Micrococcus endophyticus emerged as promising candidates because of their pronounced amylase activity. K. rosea exhibited a clear hydrolysis zone of 15 mm, while M. endophyticus demonstrated a zone of 20 mm, reflecting their efficiency in starch degradation. These two strains achieved optimal growth and produced maximum amylase at a pH of 6–7, temperatures ranging from 30 °C to 40 °C, and an incubation period of 36–72 h. Amylase activity reached its maximum efficiency at temperatures between 45 °C and 55 °C, 0.5 g/L MgCl₂ and CaCl₂, and a pH of 5–7. The amylase of M. endophyticus released 1.505 and 1.421 g/L sugar (highest activity) in acetate and phosphate buffer, respectively. Furthermore, crude amylase extracted from both isolates was used effectively in the dough leavening process, underscoring their applicability in the food industry. This study underscores the potential of K. rosea and M. endophyticus as novel sources of amylases. Full article

In this study, we constructed a linear antibody–drug conjugate (ADC), 7300-LP1003, by coupling the camptothecin derivative 095 to a linker through an ether bond. In vitro enzyme experiments indicated that LP1003 releases 095 through the action of tissue cathepsin B. Therefore, we introduced lysine pairs with different water-soluble substituents to further modify the linker and synthesized side-chain ADCs 7300-LP3004 and 7300-LP2004, modified by polysarcosine and polyethylene glycol, respectively. In vitro experiments showed that, after incubation at 55 °C in phosphate-buffered saline for 48 h, 7300-LP3004 aggregation was 45.24%, which was significantly lower than that of 7300-LP1003 (77.14%). Cell cytotoxicity assays demonstrated that the side-chain ADCs, 7300-LP3004 and 7300-LP2004, exhibited significantly higher activity (IC50 values of 39.74 nM and 32.17 nM, respectively) compared to the linear ADC and 7300-Deruxtecan (IC50 of 186.5 nM and 124.5 nM, respectively). In the subcutaneous model of SHP-77 NOD scid gamma mice, when the ADC dose was 5 mg/kg, 7300-LP3004 showed the highest tumor inhibition rate with a tumor growth inhibition (TGI) of 106.09%, which was superior to that of the positive control 7300-Deruxtecan, which had a TGI of 103.95%. In conclusion, 7300-LP3004 demonstrated strong antitumor activity and high physicochemical stability, highlighting the need for further research and development of ADC drugs. Full article

Background: This research aimed to compare the traditional in-solution digestion (inSol) and solid-phase-enhanced sample preparation (SP3) methods for salivary proteomics, with a focus on identifying mucopolysaccharidosis (MPS)-relevant proteins. Methods: Saliva samples were processed under multiple analytical conditions, including two precipitation methods (methanol or incubation with trichloroacetic acid), paired with either Rapigest or 8M urea/2M thiourea (UT) solubilization buffers. Additionally, the SP3 method was directly applied to raw saliva without pre-processing. Proteome coverage, reproducibility, digestion efficiency, and gene function were assessed. Results: The inSol method consistently provided superior proteome coverage, with trichloroacetic acid precipitation and Rapigest buffer yielding 74 MPS-relevant proteins, compared to 40 with SP3 MeOH UT. Both methods showed high digestion efficiency, particularly with Rapigest buffer, achieving over 80% full cleavage across conditions. Functional analysis revealed broad similarities, with protocol-specific impacts on protein classes and cellular components. Conclusions: This study is the first to compare SP3 and in-solution digestion for salivary proteomics, emphasizing the importance of method selection to address matrix-specific challenges. The results highlight the robustness of inSol for comprehensive proteome profiling and SP3′s potential for streamlined clinical workflows, offering valuable insights into optimizing salivary proteomics for biomarker discovery in MPS and other diseases. Full article

Levansucrases are key enzymes responsible for the synthesis of β-2,6-linked fructans, found in plants and microbes, especially in bacteria. Levansucrases have been applied in the production of levan biopolymer and fructooligosaccharides (FOSs) using sucrose as a substrate as well as in reducing sugar levels in fruit juice. As a result, levansucrases that are active at low temperatures are required for industrial applications to maintain product stability. Therefore, this work firstly reports the novel cold-active levansucrase (SacBPk) isolated from a sucrolytic bacterial strain, P. koreensis HL12. The SacBPk was classified into glycoside hydrolase family 68 subfamily 1 (GH68_1) and comprised a single catalytic domain with the Asp104/Asp267/Glu362 catalytic triad. Interestingly, the recombinant SacBPk demonstrated cold-active levansucrase activity at low temperatures (on ice and 4–40 °C) with the highest specific activity (167.46 U/mg protein) observed at 35 and 40 °C in 50 mM sodium phosphate buffer pH 6.0. SacBPk mainly synthesized levan polymer as the major product (129 g/L, corresponding to 25.8% of total sugar) with a low number of short-chain FOSs (GF2–4) (12.8 g/L, equivalent to 2.5% of total sugar) from 500 g/L sucrose after incubating at 35 °C for 48 h. These results demonstrate the industrial application potential of SacBPk levansucrase for levan and FOSs production. Full article