Mechanism Of Oxidation Research Articles

The Beneficial Effects of Curcumin on Lipids: Possible Effects on Dyslipidemia-induced Cardiovascular Complications.

Dyslipidemia and altered lipid metabolism are closely involved in the pathogenesis and clinical manifestation of many metabolic and non-metabolic diseases. Therefore, mitigation of pharmacological and nutritional factors together with lifestyle modifications is paramount. One potential nutraceutical exhibiting cell signaling and lipid-modulating properties implicated in dyslipidemias is curcumin. Specifically, recent evidence suggest that curcumin may improve lipid metabolism and prevent dyslipidemia-induced cardiovascular complications via several pathways. Although the exact molecular mechanisms involved are not well understood, the evidence presented in this review suggests that curcumin can provide significant lipid benefits via modulation of adipogenesis and lipolysis, and prevention or reduction of lipid peroxidation and lipotoxicity via different molecular pathways. Curcumin can also improve the lipid profile and reduce dyslipidemia- dependent cardiovascular problems by impacting important mechanisms of fatty acid oxidation, lipid absorption, and cholesterol metabolism. Although only limited direct supporting evidence is available, in this review we assess the available knowledge regarding the possible nutraceutical effects of curcumin on lipid homeostasis and its possible impacts on dyslipidemic cardiovascular events from a mechanistic viewpoint.

Unveiling the mechanism of thorough electrocatalytic oxidation of monoethanolamine by ultramicroelectrode and fast-scan cyclic voltammetry

The electro-oxidation of monoethanolamine (MEA) is relevant from fundamental and applied, viz. sewage treatment, fuel cell and electrochemical water splitting, perspectives. Understanding mechanistic details of thorough electrocatalytic oxidation of MEA is of param ount importance for the rational design of high-performing electrocatalysts. Herein, in order to easily and quickly quantify the electro-oxidation degree of MEA and therefore assist the mechanism study, the present paper proposes an innovative strategy for determining the number of electron transferred in the electro-oxidation of intermediates, with the aid of ultramicroelectrode (UME) and fast-scan cyclic voltammetry (CV). Deconvolutions on two fast-scan CV oxidation peaks, one of which is originated from the electro-oxidation of MEA to glycine intermediate and the other is the further electro-oxidation of intermediate, reveal that the number of electron transferred in the electro-oxidation of glycine intermediates is merely 3.04±0.75 on pure Au catalyst, indicating the incomplete conversion of glycine to ammonia and glyoxylate ion. The introduction of trace Ni into Au electrocatalyst can remarkably boost the number of electron transferred, an indication of more thorough electro-oxidation of glycine intermediates and thereby more bio-friendly degradation products. Theoretical calculations based on the density functional theory elucidate that such increase in electro-oxidation degree is due to the stronger adsorption of glycine intermediates on AuNi binary sites than on sole Au sites.

Research on the oxidation process of micro-boron below the melting point temperature

The ignition and combustion characteristics of boron and the energy release rate of boron-based fuel are closely related to the surface oxide layer of boron. To understand the oxidation process of boron below its melting point, the slow oxidation process of boron under different temperature and humidity storage conditions and the fast oxidation process under high-temperature conditions were investigated. The results indicate that the surface oxide layer mainly comprises boron (B), boron suboxides (BxOy), and boron trioxide (B2O3). When the degree of oxidation is low, BxOy and B are the main components. After deep oxidation, BxOy almost disappeared. The thickness and composition of the surface oxide layer vary with changes in environmental temperature, humidity, and aging time. The effect of temperature increase on the growth rate of the oxide layer thickness is significantly greater than that of moisture. With the prolongation of boron aging treatment time, the onset, end, and maximum weight gain rate temperatures of the boron thermal oxidation process all slightly decrease. As the ambient temperature increases, the formation of the oxide layer transitions from a unidirectional diffusion mechanism of oxygen into the interior of boron particles to a bidirectional diffusion mechanism of molten oxide and oxygen.

Mechanism of antimony oxidation and adsorption using immobilized Klebsiella aerogenes HC10 in soil

Klebsiella aerogenes HC10 is one of the few strains isolated from contaminated soil that efficiently oxidizes Sb. However, the sensitivity of microorganisms to environmental conditions limits Sb-oxidizing bacteria applications in soil remediation. Immobilizing Sb-oxidizing bacteria is a promising strategy to improve colonization rates and microorganism inefficiencies and to strengthen bioremediation in Sb-contaminated soil. This study evaluated the feasibility of an immobilization approach to enhance Sb oxidation and the remediation performance of strain HC10 in soil. The results indicated that a mixed matrix of polyvinyl alcohol and sodium alginate as fixed carriers provided a porous microstructural environment conducive to HC10 colonization and proliferation. Sb(III) concentration was reduced by 9.8 mg/L. The total Sb decreased by 3.8 mg/L by immobilized HC10 after 7 d. Key metabolites involved in Sb oxidation and adsorption were significantly upregulated. In soil, immobilized HC10 removed 48.68 % and 61.74 % of water-extractable and citric acid-extractable Sb(III), respectively. Some well-crystallized (hydr)oxide Sb fractions binding to the mineral surface were transformed into the mineral lattice form, creating an inner-sphere complex that effectively immobilized Sb. Immobilized HC10 enhanced hydrogen peroxidase, urease, and sucrase activities related to soil antioxidants and nutrient cycling. Immobilized HC10 promoted the proliferation of indigenous bacteria, which emerged as the dominant bacterial community with the potential for Sb oxidation. The ars operon genes associated with Sb resistance and transport were significantly expressed in HC10 treatments, providing a crucial basis for colonization in the soil. These results highlight the potential of immobilized Sb-oxidizing bacteria for enhanced bioremediation of Sb-contaminated soil.

Detection of the antipsychotic drug cariprazine using a low-cost MIP-based electrochemical sensor and its electrooxidation behaviour

Cariprazine (CAR), a member of the class of atypical antipsychotics, is a widely utilized pharmaceutical agent. Therefore, analytical techniques for its detection in real samples must be established, both for the purposes of treatment and for the advancement of the pharmaceutical industry. In recent years, there have been notable advancements in the development of sensor-based technologies, particularly those employing electrochemical detection for the rapid and precise analysis of a range of pharmaceuticals. Accordingly, the present study initially examined the voltammetric behaviour of CAR using an unmodified glassy carbon electrode (GCE). The mechanism of electrochemical oxidation was proposed, and subsequently, an electrochemical method for the determination of CAR was applied, which demonstrated a detection limit of 6.4 × 10−7 M (0.64 µM) and a broad linear dynamic range (2.5 × 10−6–5.0 × 10−5 M; 2.5–50.0 µM). Subsequently, a molecularly imprinted recognition system was fabricated via a simple one-step electrochemical polymerization process, using CAR as the template molecule and 3-aminophenylboronic acid as the functional monomer. A systematic optimization of the parameters influencing the sensor response was conducted. Upon identifying the optimal conditions, the MIP/GCE demonstrated a commendable sensitivity (177.25 µA/µM) and a detection limit of 1.27 × 10−14 M (12.7 fM) in a linear range from 1.0 × 10−13 to 2.5 × 10−12 M (0.1–2.5 pM). Subsequently, the sensor was effectively utilized to determine CAR in capsule and commercial serum samples, yielding satisfactory recovery values.

Recent modification, mechanisms, and performance of zinc oxide-based photocatalysts for sustainable dye degradation

The textile industry, which has crucial social and economic roles, faces sustainability challenges due to the generation of abundant wastewater contaminated by dyes. A wide range of photocatalysts has been developed to degrade dyes to reduce chemical and energy consumption in the treatment of dye-contaminated wastewater. This review mainly focuses on the photocatalytic mechanism and past studies of low-cost zinc oxide (ZnO) photocatalysts with various dopants to degrade dye degradation performance. Unlike previous reviews, this review delves deeply into property changes and possible mechanisms of ZnO-based photocatalysts induced by various dopants. ZnO-based photocatalysts are commonly doped with other semiconductors, metals, and non-metallic dopants. Semiconductor dopants such as titanium dioxide (TiO2), graphitic carbon nitride (g-C3N4), and iron oxide (Fe2O3) significantly modify the band gap and broaden the spectral response range of ZnO photocatalysts. Metallic dopants, including silver (Ag), copper (Cu), and cobalt (Co), introduce plasmonic effects that enhance the electrical conductivity and photocatalytic activity. Conversely, non-metallic dopants, especially carbon nanomaterials and sulfur (S), enable the adjustment of the band gap and surface properties of ZnO. Photocatalytic coating and thin films should be developed for photocatalyst reuse in future works.

Oxidative Stress Mechanism by Gold Compounds: A Close Look at Total ROS Increase and the Inhibition of Antioxidant Enzymes.

The antitumor activity of various gold compounds is a promising field of investigation, attracting researchers seeking potential clinical candidates. To advance this research, they explore the complex mechanisms of action of these compounds. Since the discovery of the strong inhibition of thioredoxin reductase by auranofin, the primary mechanism explored has been the inhibition of this enzyme. This inhibition disrupts the redox balance in cells, promoting oxidative stress and triggering cell death. In this review, we analyzed studies from the past decade that measured cellular ROS increase and examined the coordination structures of gold compounds. We also correlate ROS increase with the inhibition of redox-regulating enzymes, thioredoxin reductase, and glutathione reductase, to elucidate the relationship between these cellular effects and chemical structures. Our data compilation reveals that different structures exhibit varying efficacy: some significantly increase ROS production and inhibit thioredoxin reductase or glutathione reductase, while others elevate ROS levels without affecting these target enzymes, suggesting alternative mechanisms of action. This review consolidates critical evidence, enhancing our understanding of the mechanisms by which these gold complexes act and providing valuable insights for developing new therapeutic strategies against tumor cells.

Electrochemical Oxidation of UV Filters: A First-Principles Molecular Dynamics Study.

A theoretical model is proposed to study the oxidation mechanisms of the organic UV filters BP3 and BP4 during electrochemical water treatment utilizing Car-Parrinello molecular dynamics. Factors such as the amount of solvent to be included and how to design the system with the least possible intervention are discussed. The proposed model consist of the optimization of the geometries by density functional theory methods, the equilibration of the structure immersed in a water box, the inclusion of the reactive species, and the analysis of the reaction energies of each reaction pathway. The ab-initio molecular dynamics simulations lead to several products, and some trends can be identified, in accordance with the well-known reactivity rules of organic chemistry. The products proposed in this work are intermediates in longer oxidative pathways.

Self-sacrificing and self-supporting biomass carbon anode–assisted water electrolysis for low-cost hydrogen production

Electrooxidation of renewable and CO2-neutral biomass for low-cost hydrogen production is a promising and green technology. Various biomass platform molecules (BPMs) oxidation assisted hydrogen production technologies have obtained noticeable progress. However, BPMs anodic oxidation is highly dependent on electrocatalysts, and the oxidation mechanism is ambiguous. Meanwhile, the complexity and insolubility of natural biomass severely constrain the efficient utilization of biomass resources. Here, we develop a self-sacrificing and self-supporting carbon anode (SSCA) using waste corncobs. The combined results from multiple characterizations reveal that the structure-property-activity relationship of SSCA in carbon oxidation reaction (COR). Theoretical calculations demonstrate that carbon atoms with a high spin density play a pivotal role in reducing the adsorption energy of the reactive oxygen intermediate (*OH) during the transition from OH- to *OH, thereby promoting COR. Additionally, the HER||COR system allows driving a current density of 400 [Formula: see text] at 1.24 V at 80 °C, with a hydrogen production electric consumption of 2.96 kWh Nm-3 (H2). The strategy provides a ground-breaking perspective on the large-scale utilization of biomass and low-energy water electrolysis for hydrogen production.

Impact of Citrate on Mitigating Iron Mediated Polysorbate 80 Degradation in Biotherapeutic Formulation Placebos.

Polysorbate 80 (PS80), a widely used polymeric surfactant in biotherapeutic formulation, possesses a unique structural composition that effectively prevents protein aggregation in highly concentrated protein drug formulations. However, PS80 is susceptible to hydrolysis, due to the presence of fatty acid esters that can be enzymatically hydrolyzed, The unsaturated bonds in the fatty acids are prone to oxidative degradation when exposed to air, especially in the presence of transition metals such as iron and copper, which may be introduced during production and purification processes or from contamination in raw materials used in drug formulation. The degradation of PS80, particularly through metal-mediated oxidative degradation, poses a significant challenge for the industry. Among the identified trace metals, iron plays a crucial role as the redox reaction between ferrous ion (Fe(II)) and ferric ion (Fe(III)) generates radicals that initiate the degradation process. In order to investigate the impact of iron on PS80 degradation and understand the mechanism of iron-catalyzed oxidation, we utilized charge-reduction mass spectrometry and two-dimensional ion density mapping technologies to characterize the degradation of PS80. This method has proven to be a convenient and effective tool for the quick and detailed profiling of PS80, allowing for visual monitoring and examination of the changes that reflect the difficult-to-identify and easy-to-miss oxidized species of PS80. Additionally, a high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry method was developed for the separation and measurement of Fe(II) and Fe(III). Through this investigation, we determined that the involvement of Fe(II)/Fe(III) in PS80 degradation is a temperature dependent process. Furthermore, we found citrate not only promotes the conversion of Fe(II) to Fe(III), but it also chelates Fe(III) and prevents its reduction to Fe(II), thus inhibiting the initiation of the PS80 degradation. Therefore, the addition of citrate can be a crucial ingredient for controlling the degradation of PS80 in biologic drug substances and products. Overall, this investigation has provided valuable insights to enhance product stability, optimize processes, and ensure the quality of formulations containing PS80.

Pathomic and bioinformatics analysis of clinical-pathological and genomic factors for pancreatic cancer prognosis.

Pancreatic cancer exhibits a high degree of malignancy with a poor prognosis, lacking effective prognostic targets. Utilizing histopathological methodologies, this study endeavors to predict the expression of pathological features in pancreatic ductal adenocarcinoma (PAAD) and investigate their underlying molecular mechanisms. Pathological images, transcriptomic, and clinical data from TCGA-PAAD were collected for survival analysis. Image segmentation using unsupervised machine learning was employed to extract features, perform clustering, and establish models. The prognostic value of pathological features and associated clinical risk factors were evaluated; the correlation between pathological features and molecular mechanisms, gene mutations, and immune infiltration was analyzed. By clustering 45 effective pathological features, we divided PAAD patients into two groups: cluster 1 and cluster 2. Significant associations with poor prognosis were found for cluster 2 in both the training group (n = 113) and validation group (n = 75) (p = 0.006), with pathological stages II-IV identified as potential synergistic risk factors (HR = 2.421, 95% CI = 1.263-4.639, p = 0.008). Subsequently, through multi-omics correlation analysis, we further revealed a close association between cluster 2 and the oxidative phosphorylation mechanism. Within the cluster 2 group, 28 oxidative phosphorylation genes exhibited reduced expression, CDKN2A gene mutations were upregulated, and there was significant downregulation of Tregs infiltration and related immune gene expression. The pathomic model constructed using machine learning serves as a valuable prognostic target for PAAD. The histopathological features cluster 2 are closely associated with the downregulation of oxidative phosphorylation levels and Tregs immune infiltration.

Ruthenium-Substituted Polyoxoanion Serves as Redox Shuttle and Intermediate Stabilizer in Selective Electrooxidation of Ethylene to Ethylene Glycol.

The high carbon intensity of present-day ethylene glycol (EG) production motivates interest in electrifying ethylene oxidation. Noting poor kinetics in prior reports of the organic electrooxidation of small hydrocarbons, we explored the design of mediators that activate and simultaneously stabilize light alkenes. A ruthenium-substituted polyoxometalate (Ru-POM, {Si[Ru(H2O)W11O39]}5-) achieves 82% faradaic efficiency in EG production at 100 mA/cm2 under ambient conditions. Via the union of in situ spectroscopic techniques, electrochemical studies, and density functional theory calculations, we find evidence of a two-step oxidation mechanism: Ru-POM first undergoes electrochemical oxidation to the high valent state, activating ethylene via partial oxidation and forming an intermediate complex; this intermediate complex then migrates to the anode where it undergoes further oxidation to produce EG. The Ru-POM-mediated electrocatalytic system reduces the projected energy consumption required in EG production, requiring 9 GJ per ton of EG (and accompanied by 0.04 ton H2 coproduction), compared to 20-30 GJ/ton in typical prior processes.

Regulation of Oxide Pathway Mechanism for Sustainable Acidic Water Oxidation.

The advancement of acid-stable oxygen evolution reaction (OER) electrocatalysts is crucial for efficient hydrogen production through proton exchange membrane (PEM) water electrolysis. Unfortunately, the activity of electrocatalysts is constrained by a linear scaling relationship in the adsorbed evolution mechanism, while the lattice-oxygen-mediated mechanism undermines stability. Here, we propose a heterogeneous dual-site oxide pathway mechanism (OPM) that avoids these limitations through direct dioxygen radical coupling. A combination of Lewis acid (Cr) and Ru to form solid solution oxides (CrxRu1-xO2) promotes OH adsorption and shortens the dual-site distance, which facilitates the formation of *O radical and promotes the coupling of dioxygen radical, thereby altering the OER mechanism to a Cr-Ru dual-site OPM. The Cr0.6Ru0.4O2 catalyst demonstrates a lower overpotential than that of RuO2 and maintains stable operation for over 350 h in a PEM water electrolyzer at 300 mA cm-2. This mechanism regulation strategy paves the way for an optimal catalytic pathway, essential for large-scale green hydrogen production.

Nrf2-Dependent Adaptation to Oxidative Stress Protects Against Progression of Diabetic Nephropathy.

Aims: Adaptation to oxidative stress is essential for maintaining protein and redox homeostasis in mammalian cells. Palmitic acid (PA) plays a central role in oxidative stress and immunoproteasome regulation in podocytes and diabetes, and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have beneficial impact on diabetes. The role of Nrf2 in adaptation to oxidative stress and regulation of immunoproteasome by PA and EPA/DHA in podocytes and diabetic kidneys is not well defined. The present study describes the effect of PA- and EPA/DHA-induced oxidative stress in regulating Nrf2/immuoproteasome pathway in a model system relevant to diabetic nephropathy (DN). Results: Short PA exposure to podocytes promotes the upregulation of antioxidant proteins and immunoproteasome mediated by Nrf2, leading to acute transient oxidative stress adaptation. Both short- and long-term incubation of EPA or DHA in podocytes induced oxidative stress and activation of Nrf2, causing persistent oxidative stress adaptation. Long PA exposure to podocytes decreased the Nrf2 activity, and EPA/DHA attenuated these effects of PA. In db/db mice, feeding of EPA/DHA-rich fish oil increased oxidative stress in kidneys and induced renal cortical Nrf2 nuclear translocation and immunoproteasome overexpression, inhibiting the progression of DN. Innovation and Conclusion: We demonstrate an oxidative stress adaptation mechanism by PA and EPA/DHA regulated by Nrf2 in podocytes and kidneys of type 2 diabetes. This work provides an important insight into the pathogenetic mechanisms of DN by PA-induced oxidative stress. We conclude that activation of Nrf2-immunoproteasome signaling pathway by EPA/DHA plays a crucial role in abrogating the proteotoxic stress in DN. Antioxid. Redox Signal. 00, 000-000.

Oxygen Plasma Triggered Co−O−Fe Motif in Prussian Blue Analogue for Efficient and Robust Alkaline Water Oxidation

AbstractIn the context of oxygen evolution reaction (OER), the construction of high‐valence transition metal sites to trigger the lattice oxygen oxidation mechanism is considered crucial for overcoming the performance limitations of traditional adsorbate evolution mechanism. However, the dynamic evolution of lattice oxygen during the reaction poses significant challenges for the stability of high‐valence metal sites, particularly in high‐current‐density water‐splitting systems. Here, we have successfully constructed Co−O−Fe catalytic active motifs in cobalt‐iron Prussian blue analogs (CoFe‐PBA) through oxygen plasma bombardment, effectively activating lattice oxygen reactivity while sustaining robust stability. Our spectroscopic and theoretical studies reveal that the Co−O−Fe bridged motifs enable a unique double‐exchange interaction between Co and Fe atoms, promoting the formation of high‐valence Co species as OER active centers while maintaining Fe in a low‐valence state, preventing its dissolution. The resultant catalyst (CoFe‐PBA‐30) requires an overpotential of only 276 mV to achieve 1000 mA cm−2. Furthermore, the assembled alkaline exchange membrane electrolyzer using CoFe‐PBA‐30 as anode material achieves a high current density of 1 A cm−2 at 1.76 V and continuously operates for 250 hours with negligible degradation. This work provides significant insights for activating lattice oxygen redox without compromising structure stability in practical water electrolyzers.

Ambient air pollution exposure and incidence of cataract surgery: The prospective 3City-Alienor study.

Cataract, the leading cause of blindness worldwide, is a multifactorial disease involving oxidative stress mechanisms. The aim of our study was to investigate the relationship between air pollution exposure and the incidence of cataract surgery. The 3C-Alienor study is a population-based cohort of residents of Bordeaux, France, aged 65 years or more, recruited in 1999-2000 and followed every 2-3 years until 2017. Cataract surgery was self-reported and checked at slit-lamp by trained professionals. Average air pollution exposure (particulate matter ≤2.5 μm (PM2.5), black carbon (BC), nitrogen dioxide (NO2)) in the 10 years preceding baseline was estimated at the participants' geocoded residential address, using temporally adjusted land use regression. Associations of 10-year average air pollution exposure with incidence of cataract were estimated using Cox proportional hazard models adjusted for confounders. The study included 829 subjects without cataract surgery prior to inclusion; the mean age at inclusion was 72.6 years (standard deviation (SD): 4.2) and 61% were women. The median (Interquartile-range (IQR)) follow-up duration was 14.1 (6.4) years during which 507 participants underwent cataract surgery. Exposure to a concentration ≥40 μg/m3 of NO2 (the current regulatory limit value in Europe) was associated with incident cataract surgery (HR = 1.46, CI (1.16, 1.84), p = 0.001). No statistically significant association was found with PM2.5 and BC. Long-term exposure to a NO2 concentration ≥ 40 μg/m3 was associated with an increased incidence of cataract surgery. Complying with current European air pollution standards could reduce cataract surgery costs and improve population quality of life.

Insights into Catalytic Oxidative Reaction Mechanisms of Pentane on the Ru(0001) Surface

Insights into Catalytic Oxidative Reaction Mechanisms of Pentane on the Ru(0001) Surface

Formation of Both Free Hydroxyl Radicals and Surface Oxygen During Catalytic Ozonation by Single-Atom Iron: An Overlooked Pollutant-Dependent Oxidation Mechanism

Formation of Both Free Hydroxyl Radicals and Surface Oxygen During Catalytic Ozonation by Single-Atom Iron: An Overlooked Pollutant-Dependent Oxidation Mechanism

Mass Spectrometry-Based Platforms for Protein Lipoxidation Profiling.

Lipid peroxidation, occurring through enzymatic or non-enzymatic processes, generates lipid-derived electrophiles (LDEs), which can covalently modify nucleophilic amino acid residues in proteins, a process known as protein lipoxidation. This modification can alter protein structure and function, either causing damage or regulating signalling pathways. Identifying the protein targets and specific lipoxidation sites provide important clues for unveiling the oxidative stress-related protein interaction network and molecular mechanisms of related diseases. In this review, we present a detailed overview of recent advances in protein LDE modification profiling, with a focus on mass spectrometry (MS)-based chemoproteomic platforms for global protein lipoxidation profiling.

Stenosing Crohn’s disease patients display gut autophagy/oxidative stress imbalance

In this pilot study, we assessed the role of autophagy in Crohn’s Disease (CD), particularly in patients with a stenosing phenotype. Through the analysis of biopsied specimens from 36 patients, including 11 controls and 25 CD patients, categorized into inflammatory and stenosing groups, we identified a significant reduction in the autophagosomal marker Lc3b-II in patients with active inflammation and stenosis. This was paralleled by an increase in oxidative stress markers, including sNOX2-dp and H2O2, and a decrease in the antioxidant capacity measured by HBA, suggesting an imbalance in autophagy and oxidative stress mechanisms. Additionally, our findings show a correlation between autophagy markers and oxidative stress levels, indicating that autophagy dysfunction may play a pivotal role in CD and in the progression of a stenosing disease phenotype, by failing to eliminate detrimental molecules and pathogenic bacteria, thereby promoting fibrosis. This study is the first to demonstrate in vivo autophagy inhibition in stenosing CD patients and suggests that stimulating autophagic processes could offer a new avenue for the prevention and treatment of intestinal fibrosis in CD. Our results highlight the importance of exploring the interactions between autophagy, the fibrotic process, and the inflammatory cascade, opening avenues for potential therapeutic interventions in CD management.