Search Results (272)

Protein phosphorylation is a crucial post-translational modification that regulates protein activity, cellular signaling, transcriptional regulation, and cell cycle control. However, the analysis of phosphoproteins in biological samples is often compromised by complex sample matrices and interference from high-abundance proteins. While the top-down phosphoproteomics strategy enables comprehensive analysis of post-translational modifications based on intact proteins, its requirement for higher protein purity due to low protein ionization efficiency poses stern challenges. Consequently, developing appropriate enrichment methods for phosphoproteins in practical samples becomes essential. Immobilized metal ion affinity chromatography (IMAC) represents a common strategy for phosphorylated protein separation and enrichment. Among metal ions, Ti⁴⁺ has gained widespread application as IMAC chelating ligands due to its capacity to form multiple coordination networks and its high selectivity for phosphorylated protein enrichment, leveraging the strong chelating ability of phosphate groups toward metal ions. This paper presents the design and preparation of a novel magnetic Ti-IMAC nanocomposite, MNP@MPTMS–VPA–Ti(IV). The material is modified with phosphate groups via facile thiol-ene click chemistry and then immobilizes Ti⁴⁺, enabling selective enrichment of intact phosphoproteins through IMAC affinity. The efficiency of enrichment was evaluated using subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for detection and analysis. This Ti-IMAC material-based magnetic solid-phase extraction (MSPE)-MALDI-TOF MS protocol has been successfully applied to enrich intact phosphoproteins in milk and eel mucus with high selectivity, sensitivity, and suitability. Full article

To achieve the high-value utilization of pine nut resources, a novel zinc supplement was developed in this study. Pine nut protein was enzymatically hydrolyzed to prepare pine nut peptides (PP), which were subsequently chelated with zinc ions to form pine nut peptide–zinc chelate (PZn). Under optimized conditions, the zinc chelation rate of PZn reached 60.18 ± 1.77%. Peptidomic analysis revealed that PZn is composed of a select group of peptides predominantly characterized by low molecular weight (80.65 ± 1.47% < 1 kDa) and enrichment in aspartic acid, glutamic acid, and cysteine, indicating a self-selective chelation process. Comprehensive characterization via multiple techniques confirmed that zinc ions coordinate with carboxyl, hydroxyl, and thiol groups on these peptides, leading to charge neutralization, disruption of hydrogen-bonding networks, and peptide aggregation. Furthermore, bioactivity prediction of the PZn-constituting peptides revealed high intrinsic antioxidant potential, which corroborated the experimental results, showing that PZn exhibited significantly enhanced radical scavenging capacity compared to PP. These findings demonstrate that PZn possesses excellent zinc-binding capability and antioxidant activity, suggesting its potential as a novel zinc supplement, with its efficacy rooted in its specific molecular composition. Full article

Our main goal was to determine whether the accumulation of Cd and Cu is harmful for L. perenne or whether this plant can be used in the bioremediation, e.g., of wastewaters or contaminated soils. The IC50 values (concentration at which the tested parameter is inhibited to 50% against the control) for root and shoot inhibition after 14 days showed that Cu, as an essential element for plants, was more toxic than Cd. The translocation factor (TF), which refers to metal transport from the root to the shoot, did not exceed values of 0.228 and 0.353 for Cd and Cu, respectively, indicating their accumulation mostly in the roots rather than in the shoots. The protein thiol (-SH) groups as a parameter of the increased level of reactive oxygen species did not confirm the significantly higher level of oxidative stress for Cu, which is a redox-active cation. We confirmed a statistically significant positive correlation between -SH groups and chlorophyll a (r = 0.79; p < 0.05) and chlorophyll b (r = 0.84; p < 0.01) in the presence of Cd. We concluded that bioaccumulation of the tested metals occurred mostly in the roots, and the photosynthetic pigment content in the shoots was not significantly impaired by the increased presence of Cd or Cu in the shoots. Therefore, we suggest L. perenne as a suitable candidate for the phytomining or phytoextraction of metals, mostly from wastewater, in cooperation with other plant hyperaccumulators. Full article

Metal nanoparticles (NP) are increasingly used in biomedical applications. Among them, silver NPs (AgNPs) are used as active components in antibacterial coatings for wound dressings, medical devices, implants, cosmetics, textiles, and food packaging. On the other hand, AgNPs can be toxic to humans, depending on the dose and route of exposure, as agents delivering silver to cells. The cysteine residues are the primary molecular targets in such exposures, due to the high affinity of Ag⁺ ions to thiol groups. The Endothelial monocyte-activating polypeptide II (EMAP II), a cleaved C-terminal peptide of the intracellular aminoacyl-tRNA synthetase multifunctional protein AIMP1, contains five cysteines exposed at its surface. This prompted the question of whether they can be targeted by Ag⁺ ions present at the AgNPs surface or released from AgNPs in the course of oxidative metabolism of the cell. We explored the interactions between recombinant EMAP II, tRNA, and AgNPs using UV-Vis and fluorescence spectroscopy, providing insight into the effects of AgNPs dissolution kinetics on interaction EMAP II with tRNA. In addition, the EMAP II fragments binding to intact AgNPs were established by heteronuclear ¹H-¹⁵N HSQC spectra utilizing a paramagnetic probe. Structural analysis of the EMAP II reveal that the 3D structure of protein was destabilized (partially denatured) by the binding of Ag⁺ ions released from AgNPs at the most exposed cysteines. Surprisingly, this effect enhanced tRNA affinity to EMAP II, lowering its $K_d$. The course of the EMAP II/tRNA/AgNP reaction was also modulated by other factors, such as the presence of Mg²⁺ ions and TCEP, a thiol-group protector used to mimic the reducing conditions of the cell. Full article

This study investigated the effects of Auricularia auricula Polysaccharide (AAP) concentrations on the rheological and thermal properties of gluten and its subunit components. We used multiple techniques, including dynamic rheology, differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FT-IR), free thiol group analysis, and scanning electron microscopy (SEM). The results revealed that AAP increased the storage (G′) and loss (G″) modulus of gluten, glutenin, and gliadin, promoting compact elastic protein networks. DSC and free thiol group analysis demonstrated that AAP enhanced thermal stability and disulfide bond cross-linking in gluten and glutenin, but reduced thermostability and inhibited disulfide formation in gliadin. Secondary structure analysis showed 31.93% and 17.72% increases in α-helix and β-sheet content, respectively, in glutenin at 8% AAP, thereby enhancing the orderliness of the gluten structure and improving structural rigidity, while reducing gliadin’s structural order. Microscopy confirmed AAP narrowed gluten matrix pores, forming uniform honeycomb structures (though high concentrations caused disruption). In summary, AAP primarily stabilizes gluten conformation by modulating glutenin structure, thereby enhancing rheological and thermal properties. Full article

The red fluorescent protein mCherry is one of the most widely used fluorescent proteins in biology. Here, we have changed the chromophore chemistry by converting the thioether group of M66 to a thiol group through mutation to cysteine. The new variant, termed mCoral (due to its orange fluorescence hue), has similar brightness to mCherry but improved resistance to hydrogen peroxide. The variant is also responsive to pH with low and high pKa forms that have distinct spectral properties, which DFT analysis suggests is due to protonation state changes in the newly introduced thiol group, as well as the phenol group. The structure of mCoral reveals that the M66C mutation creates a space within the β-barrel structure that is filled by a water molecule, which makes new polar interactions, including the backbone carbonyl group of F65. Molecular dynamics simulations suggest that this additional water molecule, together with local solvation around the chromophore, could play a role in promoting planarity of the full conjugated system comprising the chromophore. The mCoral chromophore makes slightly more H-bonds with water than mCherry. The main water exit point for mCherry is also narrower in mCoral, providing a potential explanation for increased resistance to hydrogen peroxide. Overall, a small structural change to mCherry has resulted in a new fluorescent protein with potentially useful characteristics and an insight into the role of dynamics and water in defining the structure–function relationship in red fluorescent proteins. Full article

The study evaluated the effects of including condensed tannins (CT) from Acacia mearnsii bark in the drinking water of broilers on performance, intestinal health, and meat quality. A total of 240 male Cobb 500 chickens were distributed into three treatments: negative control (NC, without tannin), NC + 500 g/m³ of condensed tannin (CT50), and NC + 700 g/m³ of tannin (CT70), with the doses defined based on a pilot study. The experiment showed that CT50 presented better initial performance (d1–21), with greater weight gain and better productive efficiency index (PEI). But when evaluating the initial and growth phases simultaneously (d1–35), we found that birds from CT50 had better feed conversion and PEI compared to NC. When considering the total experimental period (d1–42), there was no effect of the treatment on performance. In the intestine and meat, lower levels of reactive oxygen species and lipid peroxidation were observed in CT50 and CT70 compared to NC, combined with higher levels of non-protein thiols. The CT70 treatment favored meat quality, reducing saturated fatty acids and increasing the concentration of unsaturated fatty acids, ω6, and the UFA/SFA ratio. There were no significant changes in the depth of the intestinal crypts, but the greater height of villi and number of enterocytes in the groups that consumed tannins are indicative of improved intestinal health. The thickness of the intestinal wall was lower in the chickens that consumed the condensed tannin compared to the NC. Lower concentrations of total proteins, albumin, and globulin in the serum were observed in the CT70 when compared to the NC. It is concluded that the use of tannins in the drinking water can be a viable strategy to improve the performance in the initial phase (d1–21) and meat quality of chickens. Full article

Background/Objectives: Lead, a toxic heavy metal, is widely recognized as a hazardous environmental contaminant capable of disrupting physiological homeostasis by altering stress response mechanisms and impairing pulmonary function. A comparable detrimental factor is tobacco smoking, which represents one of the most prevalent addictions worldwide. The presented study aimed to evaluate the combined impact of cigarette smoking and occupational lead exposure on selected oxidative stress biomarkers and pulmonary function parameters. Methods: 453 male employees working in a zinc smelter were recruited for participation in the study. Participants were subsequently divided into two groups: current smokers (n = 209) and former smokers (n = 244). Each group was then further subdivided according to blood lead concentration into subgroups with high (>35 μg/dL) and low (<35 μg/dL) lead levels. Venous blood samples were collected for biochemical analysis of oxidative stress parameters, including total oxidant status (TOS), malondialdehyde (MDA), protein thiol content (PSH), total antioxidant capacity (TAC), and the oxidative stress index (OSI). In addition, spirometric evaluation was conducted. Results: Former smokers demonstrated significantly more favorable oxidative stress profiles and superior spirometric outcomes compared with current smokers. No statistically significant associations were observed between lead exposure levels and either biochemical or spirometric parameters. Conclusions: Cigarette smoking appears to exert a stronger adverse influence on oxidative balance and pulmonary function than occupational lead exposure. Full article

Thiotaurine (2-aminoethane thiosulfonate) is a naturally occurring sulfur-based compound featuring a thiosulfonate group, enabling it to act as a biologically relevant donor of hydrogen sulfide (H₂S) through thiol-dependent persulfidation. H₂S levels are known to be reduced in individuals with osteoarthritis, where it plays roles in modulating inflammation, oxidative stress, and pain. This study investigated the anti-inflammatory effects of Thiotaurine in human primary chondrocytes exposed to a pro-inflammatory cytokine. Cells were pre-treated with Thiotaurine prior to stimulation with TNF-α, and the expression levels of key interleukins were assessed at both the mRNA and protein levels. TNF-α stimulation led to upregulation of IL-6, IL-8, and IL-1β, which was significantly attenuated by Thiotaurine pre-treatment. Additionally, immunofluorescence analysis showed that Thiotaurine inhibited the phosphorylation and nuclear translocation of p65, indicating suppression of NF-κB pathway activation. Persulfide detection assays confirmed an increase in intracellular persulfide levels following Thiotaurine treatment. In summary, due to its anti-inflammatory activity and ability to release H₂S, Thiotaurine emerges as a promising and potentially safe therapeutic option for osteoarthritis and other inflammation-related conditions. Full article

Background: Transcranial Magnetic Stimulation (TMS) has been employed in athletes from various sports to enhance performance; however, no data have focused on its effects in mixed martial arts (MMA). This study investigated the effects of intermittent theta-burst stimulation (iTBS), an alternative modality of TMS, on motor performance and plasma oxidative-stress biomarkers of ten male mixed martial arts (MMA) athletes. Methods: A randomized controlled trial was conducted with ten professional MMA athletes (aged 18–35 years). Participants were assigned to the experimental (iTBS) or placebo groups. Baseline and post-intervention performance were assessed using the Multiple Frequency Speed of Kick Test (MFSKT) and a Progressive Speed Kick Test (PSKT). Plasma biomarkers of oxidative stress, including thiols, total antioxidant capacity, 8-isoprostane, and carbonylated proteins, were measured before and after the performance tests in both groups. The iTBS was applied to the left primary motor cortex at an 80 motor threshold for the experimental group and at sub-threshold levels for the placebo group. A two-way ANOVA for paired groups, followed by Bonferroni post-hoc tests, were used to analyze the repeated measures, with the significance level set at p < 0.05. Results: The findings revealed significant improvements on the MFSKT [25.4 (±1.2) kicks vs. 20.8 (±1.4) kicks] and the PSKT [27.6 (±1.5) vs. 22.4 (±1.7) kicks] in the iTBS group vs. placebo, respectively. No significant differences were observed between the groups in terms of the serum redox balance biomarkers pre- and post-test, suggesting a limited impact on redox homeostasis despite performance enhancement. The placebo group showed no notable changes in either test or biomarker levels. Conclusions: These results highlight the improved physical performance in MMA athletes without altering redox biomarkers in the blood—emphasizing its applicability for neuromodulation in sports. Full article

Objectives: Gla-rich protein (GRP), a vitamin K-dependent protein, has been increasingly recognized for its dual role in modulating inflammation and inhibiting pathological calcification. Despite its emerging importance in chronic conditions, limited evidence exists regarding its behavior during acute critical illness. This study aimed to investigate the association between GRP, systemic inflammatory markers, oxidative stress (via total thiol oxidation-reduction ratio, TORR), and calcium metabolism in critically ill patients. Materials and Methods: This cross-sectional observational study included 93 critically ill patients admitted to the intensive care unit (ICU) and 60 age- and sex-matched non-critically ill volunteers. Serum GRP levels were measured using ELISA. Other biomarkers including TORR, C-reactive protein (CRP), procalcitonin (PCT), white blood cell count (WBC), immature granulocytes (IGs), and serum calcium were also analyzed. Pearson’s correlation, multivariate linear regression, and ROC analysis were performed to assess the relationships among GRP and biochemical markers, as well as their capacity to differentiate ICU patients from controls. Results: GRP, TORR, CRP, PCT, WBC, IGs, and ferritin levels were significantly elevated in ICU patients compared to the control group, whereas serum calcium levels were markedly reduced (all p < 0.05). GRP levels demonstrated moderate positive correlations with WBC (r = 0.47), neutrophils (r = 0.51), TORR (r = 0.42), CRP (r = 0.30), and IGs (r = 0.46), and a strong negative correlation with calcium (r = −0.63). In multivariate regression, TORR, CRP, WBC, IGs, PCT, and calcium levels showed significant correlations with GRP levels in univariate analysis. ROC analysis revealed that CRP had the highest discriminatory power (AUC = 0.88; 95% CI: 0.82–0.94), followed by TORR (AUC = 0.79; 95% CI: 0.71–0.86), GRP (AUC = 0.76; 95% CI: 0.68–0.84), and IGs (AUC = 0.77; 95% CI: 0.69–0.85), for distinguishing ICU patients from non-critically ill individuals. Conclusions: Our findings demonstrated that GRP is significantly associated with systemic inflammation, oxidative stress, and calcium metabolism disturbances in critically ill patients. The combined evaluation of GRP and TORR may enhance the understanding of inflammatory and oxidative mechanisms in acute critical illness. Although this study did not assess patient outcomes, these biomarkers could serve as promising candidates for future prognostic research in ICU settings. Full article

Escin is a steroidal triterpene saponin isolated from the seeds of horse chestnut (Aesculus hippocastanum L.). Due to its anti-edematous, anti-inflammatory, and venotonic properties, it is used in the treatment of chronic venous insufficiency. This study aimed to determine the effect of escin on human red blood cells (RBCs). The effect of escin on RBC hemolysis, plasma membrane fluidity, and thiol, amino, and carbonyl group levels was examined, while the conformational state of membrane proteins was also determined. Low concentrations of saponin (15–60 µg/mL) led to RBC hemolysis and increased RBC membrane fluidity, as determined using the spin probe method. Escin caused a statistically insignificant increase in thiol groups but a significant increase in amino and carbonyl groups in cell membranes. Using two spin labels that covalently bonded with thiols, we demonstrated that treatment of RBCs with escin did not affect cytoskeletal proteins or plasma membrane surface proteins. Research indicates that the main target of escin’s action is the lipid portion of the membrane, not membrane proteins. Full article

Alcohol Use Disorder (AUD) poses global health challenges, and causes hematological alterations such as macrocytosis and oxidative stress. Disruption of protein structures by alcohol and/or its metabolites may exacerbate AUDs; proteomics can elucidate the underlying biological mechanisms. This study examined the proteins differentially expressed in the cytosol and membrane fractions of erythrocytes obtained from 30 male patients with AUD, comparing them to samples from 15 age- and BMI-matched social drinkers (SDs) and 15 non-drinkers (control). The analysis aimed to identify the molecular differences related to alcohol consumption. The AUD patient subgrouping was based on mean corpuscular volume (MCV), with 16 individuals classified as having a normal MCV and 14 having a high MCV. Proteins were separated via two-dimensional(2D)-gel electrophoresis, digested with trypsin, and identified via Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (TOF) mass spectrometry (MALDI-TOF/TOF). Additionally, levels of malondialdehyde and 4-hydroxyalkenals (MDA + HAE), reduced glutathione (GSH), oxidized glutathione (GSSG), serum carbohydrate-deficient transferrin (%CDT), disialotransferrin (%DST), and sialic acid (SA) were analyzed. The results showed increased MDA + HAE and decreased total thiols in AUD patients, with GSSG elevated and the GSH/GSSG ratio reduced in the AUD MCV-high subgroup. Serum %CDT, %DST, and SA were significantly higher in AUD. Compared to the control profiles, the AUD group exhibited differential protein expression. Few proteins, such as bisphosphoglycerate mutase, were downregulated in AUD versus control and SD, as well as in the MCV-high AUD subgroup. Conversely, endoplasmin and gelsolin were upregulated in AUD relative to control. Cytoskeletal proteins, including spectrin-alpha chain, actin cytoplasmic 2, were overexpressed in the AUD group and MCV-high AUD subgroup. Several proteins, such as 14-3-3 isoforms, alpha-synuclein, translation initiation factors, heat shock proteins, and others, were upregulated in the MCV-high AUD subgroup. Under-expressed proteins in this subgroup include band 3 anion transport protein, bisphosphoglycerate mutase, tropomyosin alpha-3 chain, uroporphyrinogen decarboxylase, and WD repeat-containing protein 1. Our findings highlight the specific changes in protein expression associated with oxidative stress, cytoskeletal alterations, and metabolic dysregulation, specifically in AUD patients with an elevated MCV. Understanding these mechanisms is crucial for developing targeted interventions and identifying biomarkers of alcohol-induced cellular damage. The complex interplay between oxidative stress, membrane composition, and cellular function illustrates how chronic alcohol exposure affects cellular physiology. Full article

Background: Cirrhosis represents an advanced stage of hepatic fibrosis, and the metabolic changes resulting from liver dysfunction can cause impairment in physical capacity and quality of life. This study aimed to evaluate the effect of 12 weeks of resistance exercise on inflammatory markers, oxidative stress, physical conditioning, and quality of life in patients with liver cirrhosis. Methods: A total of 38 patients paired in the exercise (EG) and control (CG) groups participated in this study. The EG submitted to two weekly sessions of a program composed of resistance exercises. We evaluated the inflammatory markers (IL-6, IL-10, and TNF-α), oxidative stress (carbonylated protein, total thiols, enzyme superoxide dismutase, and catalase activity), body composition, handgrip strength (HGS), total volume of training, cardiorespiratory capacity (VO₂, VCO₂, and VE_peak), exercise capacity, and quality of life. Results: Regarding the EG, when comparing the follow-up period to the baseline, significant differences (p < 0.05) were found in BMI, HGS, total training volume, cardiorespiratory capacity variables, the 6 min walk test, and quality of life. Improvements were observed, without significant differences, in the inflammatory profile, oxidative stress, and one of the markers of liver function. The CG showed a significant decrease (p < 0.05) in HGS and cardiorespiratory capacity after the intervention. Conclusions: In patients with liver cirrhosis, a resistance exercise program improved physical fitness and quality of life, without adverse events. In addition, it seems that this type of training can bring some benefit to the inflammatory profile and oxidative stress of this population. Full article

(1) Background: The goal of the present study was to evaluate whether the supplementation with a multi-species probiotic in the diet of laying hens can change the microbiota and health status of the oviduct. (2) Methods: A total of 60 cages housing lightweight laying hens (36 weeks old) were randomly assigned to the following two different treatments: a control group fed a diet without probiotic, and a treatment group receiving diets supplemented with 50 g/ton of probiotics. The trial lasted for 26 weeks, after which five layers were slaughtered per treatment for oviduct (magnum) assessment, focusing on microbiome composition, oxidant and antioxidant status, and morphological analyses. Additionally, intestinal (jejunum) samples were collected to determine oxidant and antioxidant status. (3) Results: Probiotic supplementation resulted in lower counts of organisms from the RB41 order (p = 0.039) and Burkholderia genus (p = 0.017), and a total reduction in Bacillus and Corynebacterium (p = 0.050) compared to the control treatment. Genera Burkholderia (p = 0.017), Corynebacterium (p = 0.050), and Bacillus (p = 0.050) were also lower with the probiotic supplementation in relation to the control. Genera Epulopiscium (p = 0.089), Flavobacterium (p = 0.100), Ruminococcus (p = 0.089), and Staphylococcus (p = 0.100) tended to be lower in the probiotic group compared to the control. No significant differences were found between treatments for oviduct lesions. Probiotic treatment resulted in a higher protein thiol level in the intestine compared to the control (p < 0.001). However, the use of probiotics tended to reduce glutathione S-transferase levels in the oviduct compared to the control (p = 0.068). (4) Conclusions: These results suggest that dietary supplementation with probiotics can modulate the oviduct microbiota and improve the antioxidant status of laying hens, without causing tissue damage. Further research is warranted to explore the long-term implications of these changes on reproductive performance and egg quality. Full article