Search Results (257)

Protein S-nitrosylation is a selective post-translational modification in which a nitrosyl group is covalently attached to the reactive thiol group of cysteine, forming S-nitrosothiol. This modification plays a pivotal role in modulating physiological and pathological cardiovascular processes by altering protein conformation, activity, stability, and other post-translational modifications. It is instrumental in regulating vascular and myocardial systolic and diastolic functions, vascular endothelial cell and cardiomyocyte apoptosis, and cardiac action potential and repolarization. Aberrant S-nitrosylation levels are implicated in the pathogenesis of various cardiovascular diseases, including systemic hypertension, pulmonary arterial hypertension, atherosclerosis, heart failure, myocardial infarction, arrhythmia, and diabetic cardiomyopathy. Insufficient S-nitrosylation leads to impaired vasodilation and increased vascular resistance, while excessive S-nitrosylation contributes to cardiac hypertrophy and myocardial fibrosis, thereby accelerating ventricular remodeling. This paper reviews the S-nitrosylated proteins in the above-mentioned diseases and their impact on these conditions through various signaling pathways, with the aim of providing a theoretical foundation for the development of novel therapeutic strategies or drugs targeting S-nitrosylated proteins. Full article

MicroRNA-based regulatory mechanisms show disturbances related to oxidative stress (OS) interconnected with inflammation (IFM), as well as impairments associated with gestational diabetes (GDM). The aim of this study was to assess the diagnostic and prognostic significance of the OS/IFM-related microRNA in GDM by using peripheral blood mononuclear cells (PBMCs) and serum-derived extracellular vesicles (EVs) as biological samples. We selected the known OS/IFM-associated microRNAs miR-146a-5p, miR-155-5p, and miR-21-5p as candidates for our GDM biomarker analysis. Quantitative RT-PCR was employed for relative quantification of the selected microRNAs from paired samples of PBMCs and EVs derived from patients with GDM and healthy controls (n = 50 per group). The expression levels were analyzed for correlations with lipid and glycemic status indicators; metal ion-related parameters; serum thiol content; protein carbonyl and thiobarbituric acid-reactive substances’ (TBARS) levels; glutathione reductase (GR), Superoxide dismutase (SOD), and catalase (CAT) activity; and NRF2 expression. MiR-146a-5p and miR-21-5p were significantly upregulated in both PBMCs and EVs obtained from GDM patients. EVs-miR-21-5p showed a positive correlation with glycemic status in GDM patients, while miR-155-5p from PBMCs demonstrated correlation with iron-related parameters. The expression of selected microRNAs was found to correlate with NRF2 expression and SOD activity. The level of miR-146a-5p negatively correlated with neonatal anthropometric characteristics, while a higher level of PBMCs-miR-21-5p expression was determined in GDM patients with adverse pregnancy outcomes (p = 0.012). Our data demonstrate a disturbance of OS/IFM-microRNAs in GDM and illustrate their potential to serve as indicators of the associated OS-related changes, neonatal characteristics, and adverse pregnancy outcomes. Full article

Pituitary neuroendocrine tumors (PitNETs) are the most common intracranial tumors. Evidence suggests that these types of tumors may have high recurrence rates. In this context, the purinergic system, oxidative stress, and inflammation are important signaling pathways involved in the cancer’s pathophysiology. This study aimed to evaluate the sociodemographic and diagnostic profiles, as well as assess the purinergic signaling, immunological, and redox profiles, of patients after PitNET resection. We collected sociodemographic data and the patients’ diagnostic profiles. We also collected blood samples to analyze glycemia, triglycerides, albumin, and ATP levels. The ectonucleotidase activity was determined in peripheral blood mononuclear cells (PBMCs). In addition, we evaluated their redox and immunological profiles. There was a prevalence of gonadotropic macroadenoma derived from PIT-1 cells. We found that patients included in the PitNET group had increased glycemia, serum ATP levels, and ATP hydrolysis in PBMCs. Analyzing their immunological profiles, we found that patients had increased levels of IL-6, IL-10, and TNF, while the IL-27 level was decreased. Regarding their redox profiles, PitNET patients had increased levels of ROS and protein carbonylation. Unexpectedly, patients also showed increased levels of non-protein thiols (NPSHs), total thiols (PSHs), and ascorbic acid. Thus, the dysregulation of purinergic signaling sustained chronic inflammation and oxidative imbalance in PitNET patients for a long time after surgical resection. These data suggest that patients with PitNETs require long-term accompanying to prevent cancer recurrence prognosis. The biomarkers highlighted in this study may be good tools to help the medical approaches. Full article

Calpain-5/CAPN5 is a calcium-activated, non-lysosomal cysteine (thiol) protease. The substrate repertoire of CAPN5 is not known. Calpains catalyze limited proteolysis of their substrates, generating neo-N-termini that correspond to internal residues of their nascent substrate proteins. To identify such neo-N-termini generated by CAPN5, we employed an N-terminomics approach called TAILS (Terminal amine isotopic labeling of substrates) to quantitatively compare the N-terminal peptides detected in parental and CAPN5-deficient SH-SY5Y neuroblastoma cells. Thirty neo-N-termini corresponding to 29 protein groups and 24 unique proteins were detected to be depleted in the CAPN5^−/− cells. A subset of the identified putative substrates was further studied with CAPN5 co-immunoprecipitation, in vitro calcium-induced CAPN5 proteolysis assay, and their cellular fragmentation patterns were compared in parental and CAPN5-deficient SH-SY5Y cells. Here, we provide evidence for CAPN5-mediated proteolysis of the synaptic proteins DLGAP4, IQSEC1 and MPDZ, the neurodegeneration-related EWS, hnRNPU, TFG and UGP2, the DNA replication regulator MCM3, and the neuronal differentiation regulator LMTK1. Our data provide new relevance for neovascular inflammatory vitreoretinopathy (NIV), a progressive eye disease caused by pathogenic mutations in CAPN5. Data are available via ProteomeXchange with identifier PXD064313. Full article

Peroxiredoxins (Prxs; EC 1.11.1.15) are a group of thiol peroxidases that catalyze the detoxification of H₂O₂ and other organic hydroperoxides. The ripening of pepper (Capsicum annuum L.) fruit involves significant phenotypic, physiological, and biochemical changes. Based on the available pepper plant genome, eight PRX genes were identified and named CaPRX1, CaPRX1-Cys, CaPRX2B, CaPRX2E, CaPRX2F, CaPRX2-CysBAS1, CaPRX2-CysBAS2, and CaPRX Q. Among these, only CaPRX1-Cys was not detected in the transcriptome (RNA-Seq) of sweet pepper fruits reported previously. This study analyzes the modulation of these seven CaPRX genes during ripening and after treating fruits with nitric oxide (NO) gas. A time-course expression analysis of sweet pepper fruit during ripening revealed that two genes were upregulated (CaPRX1 and CaPRX2E), two were downregulated (CaPRX2B and PRX Q), and three were unaffected (CaPRX2F, CaPRX2-CysBAS1, and CaPRX2-CysBAS2). Gene expression was also studied in three hot pepper varieties with varying capsaicin contents (Piquillo < Padrón < Alegría riojana), showing a differential expression pattern during ripening. Furthermore, NO treatment of sweet pepper fruits triggered the upregulation of CaPRX2B and CaPRXQ genes and the downregulation of CaPRX1 and CaPRX2-CysBAS1 genes, while the other three remained unaffected. Among the CaPrx proteins, four (CaPrx2B, CaPrx2-CysBAS1, CaPrx2-CysBAS2, and CaPrx2E) were identified as susceptible to S-nitrosation, as determined by immunoprecipitation assays with an antibody against S-nitrocysteine and further mass spectrometry analyses. These findings indicate the diversification of PRX genes in pepper fruits and how some of them are regulated by NO, either at the level of gene expression or through protein S-nitrosation, a NO-promoting post-translational modification (PTM). Given that Prxs play a crucial role in stress tolerance, these data suggest that Prxs are vital components of the antioxidant system during pepper fruit ripening, an event that is accompanied by physiological nitro-oxidative stress. Full article

Background: A comparative analysis was conducted between two immunisation protocols using different amounts of protein extracts from adult Haemonchus contortus worms, purified by thiol-Sepharose chromatography (625 μg/animal vs. 200 μg/animal). These protocols involved either five or two inoculations of the immunogen, respectively. Methods: To evaluate the level of immunoprotection, animals were challenged with L3 of H. contortus two weeks after the last inoculation of the immunogen and humanely sacrificed at 8 weeks post-infection. Parasitological, biopathological, and serological parameters were monitored through the experiment. Parasite burden, abomasal-specific antibody responses, and histopathological changes were determined at the end of the trial. Results: The immunisation protocols resulted in similar reductions in cumulative faecal egg counts (60.5–64.9%) and the total worm burden (47.5–50%) compared to non-immunized (control) animals. Overall, these parasitological data showed an early recovery of the haematocrit (PCV) after challenge in the immunised groups relative to control. Similarly, levels of H. contortus-specific IgG and IgA antibodies increased in both the serum and gastric mucus of immunised groups. Conclusions: These findings represent a further step towards the potential application of this type of immunogen under field conditions, as protective responses (associated with a reduction in faecal egg output) were achieved using a simplified protocol, with lower immunogen doses and fewer inoculations required to induce immunoprotection, thereby mitigating the pathological effects of the parasite and reducing its ability to spread and infect susceptible hosts. Full article

This study systematically investigates how thiol–disulfide interactions influence the structure and mechanical properties of casein gels. Acid gels were prepared from suspensions of micellar casein (MC) powder that were heat-treated at 70 °C. Thiol groups were variably blocked with N-ethylmaleimide (NEM). The gels were characterized using stress–strain measurements, rheological analyses, and confocal microscopy. The stress–strain curves exhibited a biphasic behavior, with an initial linear elastic phase followed by a linear plastic region and a nonlinear failure zone. Compared to control samples, the addition of 100 mM NEM reduced the gel strength by 50%, while G′ and G″ increased by around 100%, unexpectedly. NEM-treated gels consist of uniformly sized building blocks coated with a whey protein layer. Strong physical interactions and dense packing enhance viscoelasticity under short deformations but reduce the compressive strength during prolonged loading. In contrast, control samples without NEM demonstrate weak viscoelasticity and increased compressive strength. The former is attributed to a broader particle size distribution from lower acid stability in the untreated gels, while the particularly high compressive strength of heat-treated gels additionally results from disulfide cross-links. The results show that thiol blocking and heating enable the targeted formation of acid casein gels with high shear stability but a low compressive strength. Full article

This work presents a novel microfabrication process that addresses the interference of thiol groups on off-stoichiometry thiolene (OSTE) surfaces with the curing of polydimethylsiloxane (PDMS) by integrating the high-performance polymer Parylene-C. The process utilizes a Parylene-C coating to encapsulate the active thiol groups on the OSTE surface, enabling precise replication of PDMS microstructures. Based on this method, PDMS micropillar arrays and microwell arrays were successfully fabricated and applied in passive plasma separation and polymorphic crystal formation, respectively. The experimental results demonstrate that the plasma-separation chip efficiently isolates plasma from whole-blood samples with varying hematocrit (HCT) levels, achieving a separation efficiency of up to 57.5%. Additionally, the microwell array chip exhibits excellent stability and controllability in the growth of salt and protein crystals. This study not only provides a new approach for microfabricating microfluidic chips, but also highlights its potential applications in biomedical diagnostics and materials science. Full article

Food security and food safety are major aspects for human and animal health, yet mycotoxins contaminate 60–80% of food crops before and after harvest, elevating the risk of chronic toxicity and cancer development. This study investigates the potential of ferulic acid (FA) as an antioxidant against mycotoxin-induced oxidative stress in Caco-2 cells exposed to aflatoxin B1 (AFB1) and ochratoxin A (OTA) for 24 and 48 h. The effects on the degree of lipid peroxidation and non-enzymatic and enzymatic mechanisms against oxidative stress were evaluated. FA appears to mitigate oxidative stress by modulating lipid and protein oxidation, decreasing the level of 4-hydroxy-2-nonenal (4-HNE), increasing superoxide dismutase (SOD) activity, and preserving thiol groups by scavenging reactive oxygen species (ROS). Additionally, the reduction in polyubiquitinated Nrf2 level, and higher SOD activity, suggest that FA stabilizes Nrf2, delaying its degradation and reinforcing its antioxidant role. These findings indicate that FA partially counteracts mycotoxin-induced oxidative damage, highlighting the need for further investigation into its long-term effects. Full article

Glyoxal (GO) is a reactive dicarbonyl derived endogenously from sugars and other metabolic reactions within cells. Numerous exogenous sources of this compound include tobacco smoking, air pollution, and food processing. GO is toxic to cells mainly due to its high levels and reactivity towards proteins, lipids, and nucleic acids. We speculate that glyoxal could be involved in erythrocyte protein damage and lead to cell dysfunction. The osmotic fragility and level of amino and carbonyl groups of membrane proteins of erythrocytes incubated for 24 h with GO were identified. The amount of thiol, amino, and carbonyl groups was also measured in hemolysate proteins after erythrocyte treatment with GO. In hemolysate, the level of glutathione, non-enzymatic antioxidant capacity (NEAC), TBARS, and activity of antioxidant enzymes was also determined. The study’s results indicated that GO increases erythrocyte osmotic sensitivity, alters the levels of glutathione and free functional groups in hemolysate proteins, and modifies the activity of antioxidant enzymes. Our findings indicate that GO is a highly toxic compound to human erythrocytes. Glyoxal at concentrations above 5 mM can cause functional changes in erythrocyte proteins and disrupt the oxidoreductive balance in cells. Full article

Keratin-made biomaterials, including feathers, are considered a protein-rich bioresource due to their intrinsic properties, including biocompatibility, biodegradability, mechanical resistance, and biological abundance. Beta-keratin exists as an insoluble stringy protein due to the high presence of disulfide cross-links, and as a result, it is mechanically stable and resistant to enzymatic digestion. Because of this, it is not easily decomposed, and this has made the application of feathers difficult. In this study, after dissolving feathers in NaOH, sodium sulfide, and 2-Mercaptoethanol (2-ME), the relative molecular mass of beta-keratin was calculated. Thin-layer chromatography was also used to display proteins with lower molecular weights. The antioxidant activities of the samples were evaluated by Fe-chelating and free radical scavenging tests with 2,2-diphenyl-1-picrylhydrazyl (DPPH). To investigate the effect of blocking thiol groups on the antioxidant activity of dissolved keratin, iodoacetamide and H₂O₂ were used. According to the three methods—(A) sodium hydroxide, (B) sodium sulfide, and (C) urea and 2-ME—used to extract and dissolve the feathers, method C caused the least change in the chemical structure of keratin molecules. Method A destroyed the primary structure of keratin and drastically reduced its molecular mass, but method B caused a drastic increase in the molecular mass from 9.6 kDa to higher masses, due to intermolecular bonds. For the keratin molecules dissolved by method C, the Fe-chelating activity was 93.18% and free radical scavenging was 77.45%. Blocking the thiol group with iodoacetamide initially reduced the free radical scavenging activity with DPPH by 42%, but blocking it with H₂O₂ did not affect this activity. Also, blocking of the thiol group did not initially affect Fe-chelating activity and free radical scavenging activity. After a kinetic study of the activities, an interesting observation was that both blocking agents had negative effects on radical scavenging activity, but had positive effects on Fe-chelating activity. This indicates the complexity of the role of disulfide bonds in keratin’s antioxidant behavior types. According to the observed antioxidant activities, it can be expected that beta-keratin extracted from chicken feathers is a suitable candidate for application in industrial, pharmaceutical, and health applications. Full article

Transforming Growth Factor-Beta 1 (TGF-β1) plays a pivotal role in the differentiation of fibroblasts into myofibroblasts, which is a critical process in tissue repair, fibrosis, and wound healing. Upon exposure to TGF-β1, fibroblasts acquire a contractile phenotype and secrete collagen and extracellular matrix components. Numerous studies have identified hydrogen peroxide (H₂O₂) as a key downstream effector of TGF-β1 in this pathway. H₂O₂ functions as a signalling molecule, regulating various cellular processes mostly through post-translational redox modifications of cysteine thiol groups of specific proteins. In this study, we used primary human skin fibroblast cultures to investigate the oxidative mechanisms triggered by TGF-β1. We analyzed the expression of redox-related genes, evaluated the effects of the genetic and pharmacological inhibition of H₂O₂-producing enzymes, and employed an unbiased redox proteomics approach (OxICAT) to identify proteins undergoing reversible cysteine oxidation. Our findings revealed that TGF-β1 treatment upregulated the expression of oxidant-generating genes while downregulating antioxidant genes. Low concentrations of diphenyleneiodonium mitigated myofibroblast differentiation and mitochondrial oxygen consumption, suggesting the involvement of a flavoenzyme in this process. Furthermore, we identified the increased oxidation of highly conserved cysteine residues in key proteins such as the epidermal growth factor receptor, filamin A, fibulin-2, and endosialin during the differentiation process. Collectively, this study provides insights into the sources of H₂O₂ in fibroblasts and highlights the novel redox mechanisms underpinning fibroblast-to-myofibroblast differentiation. Full article

The objective was to determine whether dairy cows may activate traditional and alternative inflammatory pathways by consuming a combination of a phytogenic diet (essential oil and pepper resin). Twenty pregnant Jersey cows in the final (third) lactation phase (260 days in milk) were divided into two groups: control, with no additive consumption, and test, with the addition of the phytogenic to the concentrate portion of the diet (150 mg/day/kg dry matter). Blood samples were collected on experimental days 1, 7, 14, 21, 28, 35, and 42 by coccygeal vein puncture to assess the complete blood count, serum biochemistry of levels of total protein, albumin, and globulin, as well as carbohydrate metabolism (glucose), lipid metabolism (cholesterol and triglycerides), protein metabolism (urea), activities of hepatic enzymes (gamma-glutamyl transferase (GGT) and aspartate aminotransferase (AST)), cytokine levels (interleukins IL-1β, IL-6, and IL-10), antioxidant response [thiobarbituric acid reactive substances (TBARS), reactive oxygen species (ROS), total thiol (PSH), and non-protein thiol (NPSH), and glutathione S(GST)], cholinergic system [total cholinesterase (ChE) and acetylcholinesterase (AChE)], purinergic signaling [NTPDase, 5′ectonucleotidase and adenosine deaminase (ADA)], and energetic metabolism enzymes [creatine kinase (CK), pyruvate kinase (PK), and adenylate kinase (AK)]. Productive performance was assessed through feed intake and milk production. The results revealed that the use of phytogenic compounds significantly influenced the cholinergic system and purinergic signaling associated with immunology. The reduction in cholinesterase (ChE) activity and the increase in acetylcholinesterase (AChE) activity in lymphocytes suggest the modulation of the cholinergic system, enhancing the immune response. Furthermore, the elevated activity of adenosine deaminase (ADA) in lymphocytes and platelets, together with increased ATP and ADP hydrolysis in platelets, indicates the beneficial regulation of purinergic signaling, potentially contributing to inflammatory modulation. These effects were accompanied by a lower production of pro-inflammatory cytokines (IL-1β and IL-6) and a higher production of IL-10, reinforcing an anti-inflammatory profile. The reduced leukocyte and lymphocyte counts may reflect a lower inflammatory demand, while the increased levels of NPSH and GST antioxidants suggest cellular protection. Despite these physiological changes, productive performance and milk quality remained unaffected. In summary and practical terms, including this additive in the cows’ diet benefits the cow’s health in the final third of gestation when the animal already has a reduced immune response due to advanced gestation. Full article

Polypyrrole (PPy) is cationic in its conducting form, requiring a charge-balancing counterion, or dopant. The release of bioactive dopants, driven by the reduction of PPy films, offers a route to controlled drug delivery. Thiol-terminated long chain poly (ethylene glycol) (PEG) reacts with a dodecylbenzene sulfonate (DBSA)-doped PPy, forming a dense overlayer and partially liberating DBSA via the chemical reduction of the film. The resulting PEG brush acts as a barrier to dopant diffusion from the film, but proteins have been shown to disrupt this layer, releasing the DBSA. The mechanism by which this disruption occurs has not been thoroughly investigated. In this study, dopant release from PEG-PPy composites was examined via systematic exposure to a variety of chemical stimuli, including macromolecules such as poly (ethylene imine), polyethylene glycol, and poloxamers, as well as small-molecular-weight alcohols, carboxylic acids, and amines. Dopant release was quantified by quartz crystal microbalance. Poly (ethylene imine) efficiently released DBSA, while anionic and uncharged macromolecules did not. All classes of small molecules triggered dopant release, with longer homologues magnifying the response. The mechanisms of dopant removal are dependent on the functional groups of the stimulating agent and include ion exchange and nucleophilic reduction of the polycationic backbone. Tosylate, salicylate, and penicillin dopants showed release behaviors similar to DBSA, demonstrating the generality of the PEG barrier. Full article

Cow’s milk is an important part of the human diet, primarily due to its high nutritional content, particularly proteins and fats. The processing of milk enhances its safety while modifying its composition and structure, influencing bioactive peptide release, which impacts protein quality, digestion, and allergenicity. Recently, filtered milk, which undergoes pasteurisation and microfiltration, has become available in UK supermarkets, offering a longer shelf life than pasteurised milk. This study aimed to evaluate the effects of microfiltration on the protein structure of cow’s milk, compared with pasteurisation, including analysis of the peptide profile released after in vitro gastrointestinal digestion of commercially available semi-skimmed filtered and pasteurised cow’s milk. Dynamic light scattering analysis revealed that the Z-average particle size of filtered milk was significantly (p < 0.05) larger than that of pasteurised milk across all brands. Additionally, filtered milk exhibited a significantly (p < 0.05) lower free thiol concentration compared with pasteurised milk, indicating structural modifications in proteins. Confocal laser scanning microscopy (CLSM) further highlighted heterogeneities in the distribution of fat and protein, with filtered milk samples showing increased interaction between fat globules and proteins. After gastrointestinal digestion, the numbers of peptides released from filtered milk exceeded those of peptides released from pasteurised milk by an average of 5%. These results provide new insights into the potential impact of microfiltration on the microstructure of milk. Further investigations using advanced analytical techniques are needed to assess the implications of these changes on protein bioavailability and human health. Full article