51 results about "Ros scavenging" patented technology

The invention relates to the field of gene engineering and molecular breeding, and discloses application of a ZmTCP5 gene in regulation and control of heat resistance of corn. According to the invention, three types of knockout mutants of the gene are obtained by using a CRISPR / Cas9 technology, and three overexpression materials are created. At normal temperature (25-28 DEG C), the material has no obvious phenotypic difference with a wild type; however, under high temperature stress (45 DEG C), the ZmTCP5 knockout mutant has enhanced heat resistance, reduced reactive oxygen species (ROS) accumulation, and significantly increased ROS scavenging enzyme activity, chlorophyll content and photochemical efficiency; on the contrary, the ZmTCP5 overexpression strain is more sensitive to heat stress, which indicates that the ZmTCP5 negatively regulates the heat resistance of the corn. The research provides a theoretical basis for analyzing a heat-resistant molecular mechanism of the corn, and also provides an important gene resource for creating a new germplasm of the heat-resistant corn.

The invention relates to the technical field of composite antibacterial materials, in particular to a preparation method and application of a spike rod-shaped nano composite material Cu-Fe-MOF (at) Ag. Rod-shaped Fe-MOF with biocompatibility is used as a carrier, Cu < 2 + > is introduced, and the appearance of the Fe-MOF is adjusted into a spike type, so that the antibacterial activity of the Fe-MOF is enhanced. On the basis of a soft and hard acid-base theory, by utilizing the principle that a d electron orbit of nano Ag is full electrons and is easy to coordinate with O and N atoms in Cu-Fe-MOF, the rodlike spike Cu-Fe-MOF (at) Ag composite nano material which can be adaptive to infected wound healing and intelligently adjust antibacterial activity and ROS (reactive oxygen species) removal capability is prepared through dual effects of adsorption and coordination. According to the material, the antibacterial activity of nano Ag can be improved, due to existence of coordinate bonds, the problem that Ag NPs are difficult to agglomerate and recycle can be solved, the problem of toxicity caused by accumulation of nano Ag on infected wounds is solved, and meanwhile, under the alkaline condition, conjugated bonds in MOF can cooperate with Ag to improve the ROS removal capacity.

The invention discloses an imitated intercorial nano preparation for treating acute respiratory distress syndrome, and belongs to the technical field of biological medicine. The internalized nano preparation comprises an active component, a liposome phospholipid bilayer and a target head, wherein the active component consists of an ROS (reactive oxygen species) removing drug and an anti-inflammatory drug. According to the invention, efficient and accurate targeting of the nano-preparation is realized by innovatively applying an imitated interment effect, an apoptotic cell marker is modified on the surface of the liposome, and an'eat me 'signal is released to simulate a natural biological process that macrophages clear apoptotic cells to be recognized and swallowed by alveolar macrophages, so that efficient and specific targeting is realized, and the targeting effect of the nano-preparation is improved. Meanwhile, up-regulation of PS receptor expression of macrophages at the inflammation part further enhances the targeting effect, and a new way is provided for efficient delivery of acute respiratory distress syndrome medicine preparations and remodeling of pathological microenvironment.

The invention provides a self-adaptive multifunctional hydrogel system for cardiac microenvironment regulation after myocardial infarction as well as a preparation method and application of the self-adaptive multifunctional hydrogel system, and belongs to the technical field of biomedical materials. The hydrogel system is hydrogel prepared by taking a micromolecular cross-linking agent, a polymer matrix and drug-loaded microspheres as raw materials. The hydrogel can realize staged response drug release in oxidative stress, inflammation and fibrosis microenvironments after myocardial infarction, has good injectability and self-healing property, and has the functions of ROS removal, nitric oxide release and the like. The invention provides a new strategy for tissue repair after myocardial infarction with multi-channel collaboration and accurate pathological response control.

The present invention relates to a food composition for improving obesity or cognitive function, comprising a hemp seed fermentate prepared using the Pediococcus acidilactici SRCM201591 strain. The fermentate was confirmed for radical scavenging ability, ROS scavenging ability, and AChE inhibitory ability, and found to have an antioxidant effect as a high content of polyphenol was measured. An anti-obesity effect was also confirmed through assays for triglyceride inhibition and C. elegans size reduction, and antioxidant and anti-obesity related gene regulation effects were identified following administration of the fermented extract. Accordingly, the fermentate can be advantageously used in the manufacture of food for improving obesity or cognitive function.

The application relates to kaempferol cerium nanoparticles and a preparation method and application thereof, and belongs to the technical field of biological medicines. The kaempferol cerium nanoparticles are prepared by performing coordination co-assembly on kaempferol and cerium ions in the presence of polyethylene glycol at a specific molar ratio, and the nanoparticles have uniform particle sizes and good dispersity. The nanoparticles have the anti-inflammatory activity of kaempferol and the ROS scavenging capacity of cerium ions, and show a significant synergistic effect in in-vivo and in-vitro experiments, can effectively reduce lung inflammation, inhibit oxidative stress, improve lung function, and have good biological safety, thereby providing a new drug candidate scheme for the treatment of ARDS.

The invention relates to the technical field of botany, in particular to a method for improving salt tolerance of apocynum venetum. The invention provides a molecular module AvSnRK1.2-AvNAC69-Tre with a sequence as shown in SEQ ID NO: 1 and application of the molecular module AvSnRK1.2-AvNAC69-Tre in improvement of the salt tolerance of apocynum venetum, specifically, the expression of a trehalose synthesis key gene AvTPPD can be regulated and controlled through phosphorylation modification, and a trehalose biosynthesis pathway is activated, so that the osmotic regulation capability and ROS removal efficiency are enhanced, and the salt tolerance of the apocynum venetum is remarkably improved; and molecular module typing can be rapidly carried out on highly heterozygous parent individuals, and dominant genotypes are selected as parents, so that the salt tolerance of offspring is improved.

The invention discloses an antioxidant mesoporous organic silicon self-assembled nano material as well as a preparation method and application thereof, and belongs to the technical field of nano materials and biomedicine. The method comprises the following steps: S1, preparing an ROS removal micromolecule precursor TK-NH2 through a mercaptan condensation reaction; s2, preparing antioxidant mesoporous organic silicon spheres (PMOs) by taking TK-NH2 as a raw material through a sol-gel method; and S3, preparing the antioxidant mesoporous organic silicon self-assembled nano material (PMOs (at) Cur (at) Ca) by taking the PMOs as a raw material through an anti-solvent method. The method is simple in steps, normal in temperature and pressure, low in cost and capable of achieving large-scale production; the prepared antioxidant mesoporous organosilicon self-assembled nano material can be applied to preparation of drugs for treating inflammations, especially inflammatory bone diseases, by removing ROS (reactive oxygen species), especially drugs for treating rheumatoid arthritis.

The application discloses a kind of self-assembly nanomaterial and its application in ROS clearance.The self-assembly nanomaterial of the application is denoted as TPP-FFYp-O, and its structural formula is shown as follows:TPP-FFYp-O of the application is specifically cut to generate TPP-FFY-O by alkaline phosphatase (ALP) overexpressed after entering inflammatory cell, TPP-FFY-O is enriched near mitochondrion by the targeting effect of TPP, and is self-assembled to form nanoparticle and stays in inflammatory cell by π-π stacking.Nanoparticle surface enrichment of a large number of TEMPO can convert superoxide radical anion (O2 ·‑ ) in mitochondrion into hydrogen peroxide (H2O2), and then decompose into oxygen and water, thereby significantly reducing the ROS level in inflammatory cell.This enzyme-controlled self-assembly nanoparticle improves the stability of TEMPO and the ability of ROS clearance, and has good application prospect.

The invention discloses oral composite microspheres for treating ulcerative colitis based on intestinal ROS storm pathological axis regulation as well as a preparation method and application of the oral composite microspheres, and aims to solve the problems that an existing ulcerative colitis treatment means is difficult to reverse core pathological circulation, oral antioxidant drugs and nano-enzymes are easily inactivated by gastric acid, the targeting property is poor, and the treatment effect is poor. And when the probiotics are independently used, the oxidation resistance is insufficient. The oral composite microsphere is prepared by taking clostridium butyricum spores as a biological carrier, firstly loading Ir10-KB nano-enzyme with peroxidase, superoxide dismutase and catalase like activities, and then carrying out sodium alginate-calcium ion cross-linking embedding. Anti-gastric acid protection and colon targeted release are achieved through the pH response gel characteristic of the sodium alginate, the flora regulation and mucous membrane repair effects of clostridium butyricum spores and the efficient ROS removal, mitochondrial protection and PANoptosis inhibition functions of the Ir10-KB nano-enzyme are synergistically exerted, and the synergistic treatment purpose of precise delivery-efficient oxidation resistance-micro-ecological regulation is achieved.

An arylalkylamine ferroptosis inhibitor based on an edaravone structure, and a preparation method therefor and a use thereof. The structural formula of the ferroptosis inhibitor is as shown in the following formula I, formula II, or formula III: formula I, formula II, formula III. The ferroptosis inhibitor has strong ROS scavenging ability and an inhibitory effect on lipid peroxidation, can inhibit ferroptosis caused by an ferroptosis inducer, and can reduce cerebral ischemia and nerve damage caused by cerebral ischemia, and alleviate symptoms of neurological diseases such as Parkinson's syndrome. On the basis of the ferrostain-1 / edaravone structure, the provided arylalkylamine ferroptosis inhibitor can exert a synergistic effect by means of multiple effects to treat ferroptosis-related diseases and can also expand the use of edaravone, thereby providing a candidate compound for subsequent drug research and development.

The application provides a preparation method of ROS-responsive hyperbranched polymer hydrogel and the hydrogel, and belongs to the technical field of hydrogel. Firstly, Michael addition reaction occurs between double bonds on PEGDA and amine groups of chain-like aliphatic diamine with ROS scavenging groups in the structure, and a hyperbranched polymer PBAE is obtained as a crosslinking agent; then esterification reaction occurs between methyl methacrylate and HA to obtain HAMA; finally, HAMA, AM and PBAE solution are mixed, and AHP hydrogel is formed through a one-step free radical polymerization reaction. The preparation method of the AHP hydrogel is simple, the material source is wide, the production efficiency is high, the AHP hydrogel has good mechanical properties, oxidation resistance and biocompatibility, and is easy to produce and prepare on a large scale.

The invention belongs to the field of biomedical nano materials, and discloses an ultra-small-size gold nano assembly (AuNAs) based on a micelle confinement in-situ growth strategy, a one-step preparation method and application. According to the assembly, an amphiphilic block copolymer pluronic F127 and n-dodecanethiol serve as a composite micelle template, reduction, nucleation and ordered assembly of a gold precursor are synchronously completed in a confinement space, the hydration particle size is 27.37 + / -4.95 nm, and the assembly is uniform in structure and excellent in stability. In-vitro experiments show that the assembly has efficient scavenging capacity on ABTS < + >, DPPH and OH (the scavenging rates are respectively 98.37%, 51.9% and 93.9% under the concentration of 10.0 nM), has no obvious toxicity on HK-2 cells within the concentration range of 0-20 nM, and can effectively remove excessive reactive oxygen species (ROS) in the cells. The preparation process is simple and convenient, the cost is controllable, the prepared AuNAs can be used for preparing ROS removal type cell protective agents or candidate drugs for treating oxidative stress related diseases, and the AuNAs have good industrial application prospects and clinical transformation potential.

The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to a gentiopicroside nitroxide radical derivative as well as a preparation method and application thereof. A series of gentiopicroside nitroxide radical derivatives with specific structures are prepared and synthesized through a chemical process. The derivatives exhibit good in-vitro COX-2 enzyme inhibition activity and in-vitro ROS scavenging activity. In-vivo and in-vitro experimental results show that each derivative can effectively inhibit the release of inflammatory mediators and oxidative stress during inflammation, thereby more effectively inhibiting the progress of inflammation and protecting tissues, and the derivatives have a wide application prospect in the preparation of drugs with the efficacy of preventing, relieving or treating inflammation.

The application discloses a 4-(benzo[d]thiazol-2-yl)benzene-1,2-ethylenediamine compound and a preparation method and application thereof. The structural formula of the compound is: the compound has strong ROS scavenging capacity, inhibits lipid peroxidation, can inhibit ferroptosis caused by an iron death inducer, can reduce brain ischemia and nerve damage caused by brain ischemia, and can relieve symptoms of Parkinson's syndrome and other neurological diseases. Therefore, the ferroptosis inhibitor based on the ferrostain-1 / lirilad structure provided by the application can treat diseases related to ferroptosis through multiple actions and has the potential to be developed into a drug for treating diseases related to ferroptosis. Moreover, the ferroptosis inhibitor can be used as an antioxidant fluorescent probe for high-sensitivity detection of a ferroptosis marker, lipid peroxide (PCOOH / PEOOH), can effectively reverse the ferroptosis process of cells, and has a self-indication capacity. The ferroptosis inhibitor is a solid powder, is convenient to store and use, has a simple synthesis method, high yield, low cost, and good popularization prospect.

The application discloses oil-soluble PDRN and a preparation method and application thereof. The preparation method comprises the following steps: dissolving PDRN in an alkaline solvent to obtain a PDRN solution; adding an acylation agent into the PDRN solution to perform acylation modification, and precisely controlling pH in the whole process to obtain a reaction product liquid; performing salting-out and centrifugation on the reaction product liquid, and collecting a precipitate; and performing washing and vacuum drying on the precipitate to obtain oil-soluble PDRN. Through the acylation modification strategy of precisely controlling pH in the whole process, the oil-soluble PDRN with high acylation degree, excellent oil solubility, excellent ROS scavenging capacity, high DPPH free radical scavenging rate, excellent thermal stability and excellent light stability is successfully prepared, the oil-soluble PDRN can be dissolved in an oil phase matrix, and a technical breakthrough of improving oil solubility, retaining biological activity and enhancing antioxidant activity is realized.

This invention belongs to the field of nanomaterials technology, specifically relating to the preparation of nucleic acid-guided supramolecular semiquinone radical nanoparticles and their application in tumor photothermal therapy. The nucleic acid-guided supramolecular semiquinone radical nanoparticles provided by this invention are formed by non-covalent bonding of long single-stranded DNA containing repeating G-quadruplex units with polydopamine, and can be used to prepare tumor photothermal therapy reagents. This invention improves both the photothermal conversion capacity and ROS scavenging capacity of the nanomaterials by modifying the structure of polydopamine, achieving a synergistic effect in PTT (photothermal phototherapy).

This disclosure provides a method for alleviating aluminum toxicity in hydroponic peas based on menadione-mediated reactive oxygen species (ROS) metabolism. The core components include: regulating the concentration and duration of menadione treatment to optimize the spatiotemporal accumulation pattern of ROS (superoxide anion, hydrogen peroxide) in the roots of hydroponic peas; adjusting the dynamic response of endogenous antioxidant enzyme systems (SOD, POD, APX) to enhance menadione-mediated ROS scavenging capacity; controlling the initial ROS burst based on changes in plasma membrane NADPH oxidase (Rboh) gene expression and enzyme activity to inhibit the aluminum stress oxidative signaling cascade; and adjusting the timing and frequency of menadione administration based on subcellular-scale mitochondrial and chloroplast ROS leakage rates to alleviate organ-specific oxidative damage. This approach addresses five key issues: aluminum-induced oxidative damage, cellular redox imbalance, oxidative signaling cascade, organ-specific oxidative damage, and persistent aluminum toxicity metabolic disorders.

The present application relates to the field of biological medicine and tissue engineering, and particularly relates to a nano-enzyme functionalized dynamic hydrogel as well as a preparation method and application thereof.The nano-enzyme functionalized dynamic hydrogel comprises black phosphorus nanosheets and nano-enzymes.The preparation method of the nano-enzyme functionalized dynamic hydrogel comprises the following steps: (1) synthesis of a hydrogel precursor solution; (2) preparation of biomimetic nano-enzymes; and (3) preparation of a dynamic hydrogel.In the present application, BP can maintain Ce(III) / Ce(IV) redox cycling through self-oxidation and improve the ROS scavenging capacity in the microenvironment, thereby serving as CeO x a stabilizer of nano-enzymes.In addition, the MBC obtained by wrapping BP@CeO x in the nucleus pulposus cell membrane can better achieve nucleus pulposus cell targeting, so that BP@CeO x can effectively and durably scavenge intracellular ROS.

The application discloses a preparation method of a lysozyme-nano selenium compound and application of the lysozyme-nano selenium compound in ROS removal. To mix sodium selenite solution and lysozyme uniformly, a lysozyme-sodium selenite solution is obtained, then vitamin C is added, and the mixture is uniformly mixed under the condition of light shielding and at room temperature, the pH value is adjusted to 6.5-7.5, a red solution is obtained, and dialysis is performed, so that the lysozyme-nano selenium compound is obtained. The lysozyme-nano selenium compound has the advantages of simple process, mild preparation condition, higher stability, biological safety, efficient ROS removal in egg white, and no influence on the regulation ability of the functional property, edible quality and other characteristics of the egg white, so that the deterioration problem of the egg white in the storage process is solved. The lysozyme-nano selenium compound has good application prospect in ROS removal, and can be applied to the fields of biomedical science and material science.

The application provides an injectable black phosphorus nanosheet-loaded composite hydrogel and a preparation method and application thereof. The composite hydrogel is prepared by mixing polydopamine-functionalized black phosphorus nanosheets, a 2-2.5% wt / vol sodium alginate suspension, a 1.5-2% wt / vol calcium alginate suspension and hyaluronic acid; wherein the volume ratio of the 2-2.5% wt / vol sodium alginate suspension, the 1.5-2% wt / vol calcium alginate suspension and the hyaluronic acid is 5:5:2, and the concentration of the polydopamine-functionalized black phosphorus nanosheets in the synthesized composite hydrogel is 100 μg / ml; and the application of the injectable black phosphorus nanosheet-loaded composite hydrogel in a myocardial infarction disease injection drug, through ROS scavenging to inhibit the NFκB signal pathway and reduce inflammatory reactions, the black phosphorus nanosheet-loaded composite hydrogel in the application can be used as a ROS scavenging, anti-inflammatory, anti-apoptosis, conductive and biodegradable platform for tissue engineering applications.

The invention belongs to the technical field of biomedical materials. The invention provides a nano-enzyme-loaded hydrogel compound as well as a preparation method and application thereof.The nano-enzyme-loaded hydrogel compound is prepared by taking thermo-sensitive chitosan / sodium glycerophosphate hydrogel as a material substrate, jointly loading Mn-POM nano-enzyme and a TRPA1 channel antagonist HC-030031 on the material substrate, and stirring under an ice bath condition. The nano-enzyme drug-loaded hydrogel compound prepared by the preparation method disclosed by the invention integrates in-situ forming, ROS (reactive oxygen species) removal, microenvironment response drug release and synergistic anti-inflammation.

The invention belongs to the technical field of medicinal chemistry, and particularly relates to a selenium-containing gentiopicroside nitroxide free radical derivative as well as a preparation method and application thereof. A series of selenium-containing gentiopicroside nitroxide free radical derivatives with a specific structure are prepared and synthesized through a chemical process, and the selenium-containing compound derivatives obtained by adding a selenium-containing structure on the basis of the gentiopicroside nitroxide free radical derivatives show good in-vitro ROS scavenging activity and anti-inflammatory activity; the expression level of an inflammatory medium iNOS is effectively reduced, and the compound has a wide application prospect in preparation of medicines with the effects of preventing, relieving or treating inflammation.

The invention provides a carbon dot with double functions of near-infrared two-region imaging and ROS (reactive oxygen species) removal and a preparation method of the carbon dot. The carbon dot is prepared by reacting an aniline monomer containing a p-phenylenediamine amino group and selenourea as precursors, and has a near-infrared second region (NIR-II) fluorescence emission characteristic and active oxygen (ROS) scavenging capacity.

The application discloses a chemical cross-linking preparation method and application of an anti-inflammatory and anti-oxidation anti-adhesion hydrogel, and belongs to the technical field of biological medical material preparation. The method is to cross-link and construct a gel body by using disulfide bonds and amidation reaction, so that the stability of the gel body is enhanced, the residence time of the gel body in tissues is further prolonged, the anti-inflammatory and anti-oxidation performance of the gel body is increased due to the fact that the gel body contains mercapto groups, and finally the anti-adhesion effect of the product is enhanced. The anti-adhesion hydrogel product is prepared by the chemical cross-linking method. The application of the anti-adhesion hydrogel product in preparing and treating postoperative peritoneal tissue adhesion products is proved by in-vitro ROS scavenging experiments, and the free radical scavenging rate of the anti-adhesion hydrogel prepared by the technical scheme of the application can reach more than 50%.

The proposed invention is a reactive oxygen species scavenging polymer system with biodegradable and biodurable embodiments for biomedical applications. Several embodiments are tailored for patients with high oxidative stress due to comorbidities (diabetes, obesity, peripheral neuropathy, osteoporosis, osteopenia, chronic kidney disease, neurodegenerative diseases, and cardiovascular disease). Other embodiments function independent of atypical oxidative stress to provide a biodegradable biomaterial system. Several embodiments include thiourethane synthetic schemes to create biomaterials for applications requiring increased toughness.

The application belongs to the field of functional food development and biological medicine technology, and relates to a carrier for intestinal targeting and ROS scavenging and a preparation method and application thereof. The application provides a preparation method of the carrier for intestinal targeting and ROS scavenging. Low-ester pectin is heated and dissolved, then gelatin and glycerol are added to prepare a mixed solution, microalgae are added to the mixed solution to obtain a film-forming solution, and the carrier is prepared from the film-forming solution. The application mixes activated probiotics with the film-forming solution to prepare a ROS scavenging edible film loaded with probiotics. The ROS scavenging edible film can effectively resist the adverse environment of the stomach to probiotics, so that the probiotics can successfully reach the intestine and play a role.

The invention relates to the technical field of cerebrovascular diseases, and particularly discloses a drug controlled-release nano-drug capable of penetrating through BBB and removing ROS and a preparation method of the drug controlled-release nano-drug. The drug controlled release nano drug capable of penetrating through BBB to remove ROS comprises a nano coordination polymer core responsive to ROS, the core is composed of diselenide bond bridged organic ligands and metal ions, resveratrol is loaded on the core, and mannose-polyethylene glycol targeting molecules are modified on the surface of the core; the preparation method comprises the following steps: S1, synthesizing an organic ligand containing a diselenide bond; s2, synthesizing a nano coordination polymer core; s3, loading the resveratrol; s4, synthesizing a targeting molecule; and S5, surface modification. The drug controlled release nano-drug for removing ROS through BBB can be used for efficient synergistic treatment of cerebral arterial thrombosis, and has the advantages of precise focus targeting, on-demand drug release and multiple curative effect synergy.

The invention discloses a nucleic acid drug delivery system and a preparation method and application thereof. The nucleic acid drug delivery system comprises photo-crosslinking hydrogel and cationic lipid nanoparticles, the cationic lipid nanoparticles are loaded in the photo-crosslinking hydrogel, the cationic lipid nanoparticles encapsulate siRNA for inhibiting REST gene expression, and the nucleotide sequence of the siRNA comprises the sequences shown as SEQ ID No.1 and SEQ ID No.2. The invention also discloses a preparation method of the nucleic acid drug delivery system, and the preparation method of the nucleic acid drug delivery system comprises the following steps: preparing the cationic lipid nanoparticles, after the nucleic acid drug delivery system is implanted into a spinal cord injury local tissue, cationic lipid nanoparticles encapsulating siRNA are slowly released, continuous silencing of REST genes is achieved, axon regeneration is promoted, meanwhile, the photo-crosslinking hydrogel plays a role in removing ROS, in addition, M2 polarization of macrophages can be promoted to regulate and control the immune microenvironment, and the effect of treating spinal cord injury is achieved. Therefore, nerve repair is synergistically promoted at molecular and cellular levels, double-track parallelism of'immune regulation-axon regeneration 'is realized, and the polypeptide has a wide application prospect in treatment of spinal cord injury.

The application discloses a method for improving the stability of aloe polysaccharide microsphere emulsion. The method adds a specific parameter water bath ultrasonic treatment step on the basis of the existing aloe polysaccharide microsphere emulsion preparation process. The aloe polysaccharide microsphere emulsion is placed in an ultrasonic generator, purified water is added as a conducting medium, ultrasonic waves with a frequency of 35-45 kHz and a power of 200-300 W are used for water bath ultrasonic treatment, the treatment time is 0.5-3 hours, and the ultrasonic water temperature is controlled to be 45-55 DEG C. The method can significantly reduce the microsphere particle size, increase the microsphere quantity, and improve the absolute value of the surface potential, thereby improving the long-term storage stability of the emulsion. Meanwhile, the emulsion after treatment shows stronger ATP synthesis recovery, mitochondrial membrane potential stability and ROS scavenging capacity at the cell level, and has enhanced cell protection efficacy.