Self-activation Mechanism Research Articles

Exploring the constitutive activation mechanism of the class A orphan GPR20.

GPR20, an orphan G protein-coupled receptor (GPCR), shows significant expression in intestinal tissue and represents a potential therapeutic target to treat gastrointestinal stromal tumors. GPR20 performs high constitutive activity when coupling with Gi. Despite the pharmacological importance of GPCR constitutive activation, determining the mechanism has long remained unclear. In this study, we explored the constitutive activation mechanism of GPR20 through large-scale unbiased molecular dynamics simulations. Our results unveil the allosteric nature of constitutively activated GPCR signal transduction involving extracellular and intracellular domains. Moreover, the constitutively active state of the GPR20 requires both the N-terminal cap and Gi protein. The N-terminal cap of GPR20 functions like an agonist and mediates long-range activated conformational shift. Together with the previous study, this study enhances our knowledge of the self-activation mechanism of the orphan receptor, facilitates the drug discovery efforts that target GPR20.

Reversible phase transition poly(benzyl methacrylate)/ionic liquid electrolytes for effective overheating protection in lithium batteries

Battery safety is influenced by various factors, with thermal runaway being one of the most significant concerns. While most studies have concentrated on developing one-time, self-activating mechanism for thermal protection, such as temperature-responsive electrodes, and thermal-shutdown separators, these methods only provide irreversible protection. Recently, reversible temperature-sensitive electrolytes have emerged as promising alternatives, offering both thermo-reversibility and self-protective properties. However, further research is crucial to fully understand these thermal-shutdown electrolytes. In this study, we propose lower critical solution temperature (LCST) phase behavior poly(benzyl methacrylate)/imidazolium-based ionic liquid mixtures to prepare temperature-sensitive electrolytes that provide reversible thermal shutdown protection of batteries. This electrolyte features an appropriate protection temperature (∼105°C) and responds quickly within a 1 min at 105°C, causing cells to hardly discharge as the voltage suddenly drops to 3.38 V, and providing efficient thermal shutdown protection within 30 min. Upon cooling back to room temperature, the battery regains its original performance. Additionally, the electrolyte exhibits excellent cycling stability with the capacity retention of the battery is 91.6% after 500 cycles. This work provides a viable solution for preventing batteries from thermal runaway triggered by overheating.

Double C-H Amination of Naphthylamine Derivatives by the Cross-Dehydrogenation Coupling Reaction.

A high-efficiency tandem process has been developed for the formation of two C-N bonds through a cross-dehydrogenative coupling (CDC) amination of spiro[acridine-9,9'-fluorene]s (SAFs) with amines. This method offers a strategically innovative and atom-economical approach to obtaining diamine-substituted SAFs. Notably, the approach eliminates the need for metal catalysts and other additives, relying solely on O2 as the oxidant. A self-activation mechanism has been proposed to elucidate the effective double amination in the CDC process.

Autonomous, temperature-enhanced oxidase-mimic multimodal antimicrobial film for prolonged preservation of perishable products

Confronting the escalating challenges of food waste and safety, primarily attributed to microbial-induced spoilage, this investigation unveils a cutting-edge nanozyme-based antimicrobial packaging paradigm with self-activating mechanism (CMn0.20-CS film), employing low-thermal aid to optimize the preservation of fruits and vegetables. At its core, the system amalgamates a carbon nanotube-supported Mn3O4 (CMn) filler within a chitosan (CS) film matrix. The fabrication of the CMn complex involves a dual-stage process, initially focusing on the immobilization of Mn2+ ions, followed by the wet chemical nucleation of Mn3O4 crystals. A pivotal feature of this construct is the weak acidity of CS matrix, which imparts the CMn0.20-CS film with a self-activating capacity, facilitating an optimal environment for the manifestation of oxidase-like activity. The CMn0.20-CS film exhibits a synergistic antimicrobial efficacy, derived from the augmented oxidase-mimetic and photothermal characteristics of CMn, in conjunction with the intrinsic microbial capture and killing capabilities of CS. This translates into formidable bactericidal proficiency (exceeding 99.99 % for bacteria and 99.67 % for mold), swift action (within 10 min), and a requirement for minimal auxiliary heat (56.4 ℃). Based on this, the CMn0.20-CS film significantly prolongs the shelf life of kumquats, extending its shelf life by 2.3 times. The augmented mechanical strength, improved barrier qualities, and demonstrated safety profile of the film underscore its potential for practical applications. Collectively, the CMn0.20-CS film stands out as an exceptionally suitable method for the effective preservation of perishable goods, tackling fundamental concerns in food safety and wastage mitigation.

Heterotrimeric G Protein-Mediated Signaling Is Involved in Stress-Mediated Growth Inhibition in Arabidopsis thaliana.

Heterotrimeric G protein-mediated signaling plays a vital role in physiological and developmental processes in eukaryotes. On the other hand, because of the absence of a G protein-coupled receptor and self-activating mechanism of the Gα subunit, plants appear to have different regulatory mechanisms, which remain to be elucidated, compared to canonical G protein signaling established in animals. Here we report that Arabidopsis heterotrimeric G protein subunits, such as Gα (GPA1) and Gβ (AGB1), regulate plant growth under stress conditions through the analysis of heterotrimeric G protein mutants. Flg22-mediated growth inhibition in wild-type roots was found to be caused by a defect in the elongation zone, which was partially blocked in agb1-2 but not gpa1-4. These results suggest that AGB1 may negatively regulate plant growth under biotic stress conditions. In addition, GPA1 and AGB1 exhibited genetically opposite effects on FCA-mediated growth inhibition under heat stress conditions. Therefore, these results suggest that plant G protein signaling is probably related to stress-mediated growth regulation for developmental plasticity in response to biotic and abiotic stress conditions.

Pyrolysis self-activation: An environmentally friendly method to transform biowaste into activated carbon for arsenic removal

A green method for production of activated carbon and combustible gas was introduced. Without any external reagents and gases, the H2O and CO2 produced by the pyrolysis of bamboo shoot shells were used as activators. The prepared activated carbon had good arsenic adsorption properties with the maximum adsorption capacities of 10.9 mg/g for As(III) and 16.0 mg/g for As(V). The gaseous products were mostly CO and H2, with higher heating value of 11.7 MJ/Nm3. Thermogravimetric experiments were performed in N2, H2O and CO2 atmospheres to simulate the self-activation process and investigate the self-activation mechanism. This work will help to improve the competitiveness of self-activation technology and reduce the production cost of activated carbon.

A New Pathway for CO2 Reduction Relying on the Self-Activation Mechanism of Boron-Doped Diamond Cathode.

By means of an initial electrochemical carbon dioxide reduction reaction (eCO2RR), both the reaction current and Faradaic efficiency of the eCO2RR on boron-doped diamond (BDD) electrodes were significantly improved. Here, this effect is referred to as the self-activation of BDD. Generally, the generation of carbon dioxide radical anions (CO2•–) is the most recognized pathway leading to the formation of hydrocarbons and oxygenated products. However, the self-activation process enabled the eCO2RR to take place at a low potential, that is, a low energy, where CO2•– is hardly produced. In this work, we found that unidentate carbonate and carboxylic groups were identified as intermediates during self-activation. Increasing the amount of these intermediates via the self-activation process enhances the performance of eCO2RR. We further evaluated this effect in long-term experiments using a CO2 electrolyzer for formic acid production and found that the electrical-to-chemical energy conversion efficiency reached 50.2% after the BDD self-activation process.

Capacitive dominated charge storage in supermicropores of self-activated carbon electrodes for symmetric supercapacitors

The present work demonstrates a systematic study of pore size and specific surface area (SSA) of biomass-derived carbon and the choice of electrolyte concentrations affecting charge-storage mechanism (surface controlled and diffusion controlled) and electrochemical behaviour. Porous nanocarbons derived from Caesalpinia Sappan pods were synthesized by pyrolysis at 400, 600, and 800 °C. Pyrolysis at 800 °C was found suitable for the self-activation mechanism which formed bimodal porous nanocarbons with a high SSA of 675 m2/g. A maximum specific capacitance of 261.8 F/g at 0.5 A/g in 5.0 M KOH was observed for electrode materials synthesized at 800 °C. The highlight of the study is the porous nanocarbon synthesized at 800 °C which was found to possess micropores of size 0.7–1.0 nm playing a pivotal role in enhancing capacitance. The effect of electrolyte concentration on capacitance and charge storage mechanisms was also analyzed. A diffusion-controlled self-discharge model is established for supercapacitor devices. The single cell can power a red LED for 15 min; exemplifying the sustainable strategy of the utilization of abundant bio-waste to efficient energy storage devices.

Developing self-activated lignosulfonate-based porous carbon material for ethylene adsorption

This study applied lignosulfonate as raw material to develop porous carbon for ethylene adsorption, followed by testing for this material's ethylene adsorption efficiency and capacity. The results indicated that the gas released (CH4, H2, CO, and CO2) during pyrolysis could generate a pore structure on lignosulfonate-based carbon surface, regarded as the self-activated mechanism. Pore structure formation increased pore volume and resulted in generation of various pore shapes. When carbonization temperature was less than 800 °C, specific surface area of the samples increased with temperature, and it decreased otherwise. Moreover, with increasing carbonization temperature, the samples’ carbon content increased but hydrogen content decreased. Ethylene adsorption uptake increased as carbonization temperature increased. Adsorption capacity was maximum when carbonization temperature was 800 °C. It was influenced by the pore structure, and specifically, it increased as micropore volume increased. In addition, physical adsorption was the main mechanism of ethylene adsorption for the lignosulfonate-based porous carbon, leading to efficient adsorption-desorption cycles. Finally, its application as an ethylene scavenger can potentially transcend currently available products.

Up-regulated cathepsin C induces macrophage M1 polarization through FAK-triggered p38 MAPK/NF-κB pathway

Increasing evidence indicates that in response to environmental changes, macrophages can dynamically change into two main functional phenotypes, namely M1 and M2. Depending on these different phenotypes, macrophages can produce either pro-inflammatory or anti-inflammatory factors which may affect the outcome of inflammation. Mastering the switching of M1/M2 phenotypes may provide therapeutic approaches to chronic inflammatory disease, such as atherosclerosis, rheumatoid arthritis, even the metabolic disorders. Cathepsin C (CTSC), as a member of the papain family of cysteine proteases, is a key enzyme in the activation of granule serine proteases thereby involved in modulating the inflammatory responses. Moreover, abundant expression of CTSC has been found in M1 macrophages in plaques of atherosclerosis and related to the progression of disease. However, whether CTSC can regulate macrophage activation status in inflammatory responses has not been fully investigated. In the present study, using peritoneal macrophages (PMs) and mouse macrophage cell line RAW264.7 treated with LPS and active monomer of CTSC, we found that CTSC was not only expressed in macrophages in M1 activation status, but also facilitated macrophages towards M1 phenotype, suggesting a self-activation mechanism involved in this process which may lead to a vicious circle in chronic inflammation. Then we attempted to explore the underlying molecular mechanisms of CTSC resulting in M1 activation. Focal adhesion kinase (FAK) is one of the non-receptor cytoplasmic protein tyrosine kinases, serving as an upstream mediator that leads to transcription of many pro-inflammatory factors. We found FAK expression was up-regulated at both mRNA and protein levels following CTSC stimulation, and FAK phosphorylation level was also significantly increased. The p38MAPK/NF-κB pathway, as the downstream of FAK, were also found activated in CTSC-treated macrophages, suggesting that CTSC may promote macrophage towards M1 activation status through FAK-induced p38MAPK/NF-κB signaling pathway activation. Our study provides a new therapeutic target in the treatment of chronic inflammatory diseases.

A Novel Self-Activated Mechanism for Stable Liquid Transportation Capable of Continuous-Flow and Real-time Microfluidic PCRs.

The self-activated micropump capable of velocity-stable transport for both single-phased plug and double-phased droplet through long flow distance inside 3D microchannel is one dream of microfluidic scientists. While several types of passive micropumps have been developed based on different actuation mechanisms, until today, it is still one bottleneck to realize such a satisfied self-activated micropump for the stable delivery of both single and double-phased liquid inside long microchannel (e.g., several meters), due to the lack of innovative mechanism in previous methods. To solve this problem, in this article, we propose a new self-activated pumping mechanism. Herein, an end-opened gas-impermeable quartz capillary is utilized for passive transport. Mechanism of this micropump is systemically studied by both the mathematical modeling and the experimental verifications. Based on the flow assays, it totally confirmed a different pumping principle in this paper, as compared with our previous works. The value of the overall flow rates inside the 3D microchannel is confirmed as high as 0.999, which is much more homogeneous than other passive pumping formats. Finally, this novel micropump is applied to continuous-flow real-time PCRs (both plug-type and microdroplet-type), with the amplification efficiency reaching 91.5% of the commercial PCR cycler instrument.

Small GTPase patterning: How to stabilise cluster coexistence.

Many biological processes have to occur at specific locations on the cell membrane. These locations are often specified by the localised activity of small GTPase proteins. Some processes require the formation of a single cluster of active GTPase, also called unipolar polarisation (here “polarisation”), whereas others need multiple coexisting clusters. Moreover, sometimes the pattern of GTPase clusters is dynamically regulated after its formation. This raises the question how the same interacting protein components can produce such a rich variety of naturally occurring patterns. Most currently used models for GTPase-based patterning inherently yield polarisation. Such models may at best yield transient coexistence of at most a few clusters, and hence fail to explain several important biological phenomena. These existing models are all based on mass conservation of total GTPase and some form of direct or indirect positive feedback. Here, we show that either of two biologically plausible modifications can yield stable coexistence: including explicit GTPase turnover, i.e., breaking mass conservation, or negative feedback by activation of an inhibitor like a GAP. Since we start from two different polarising models our findings seem independent of the precise self-activation mechanism. By studying the net GTPase flows among clusters, we provide insight into how these mechanisms operate. Our coexistence models also allow for dynamical regulation of the final pattern, which we illustrate with examples of pollen tube growth and the branching of fungal hyphae. Together, these results provide a better understanding of how cells can tune a single system to generate a wide variety of biologically relevant patterns.

Is IQG-607 a Potential Metallodrug or Metallopro-Drug With a Defined Molecular Target in Mycobacterium tuberculosis?

The emergence of strains of Mycobacterium tuberculosis resistant to isoniazid (INH) has underscored the need for the development of new anti-tuberculosis agents. INH is activated by the mycobacterial katG-encoded catalase-peroxidase, forming an acylpyridine fragment that is covalently attached to the C4 of NADH. This isonicotinyl-NAD adduct inhibits the activity of 2-trans-enoyl-ACP(CoA) reductase (InhA), which plays a role in mycolic acid biosynthesis. A metal-based INH analog, Na3[FeII(CN)5(INH)]·4H2O, IQG-607, was designed to have an electronic redistribution on INH moiety that would lead to an intramolecular electron transfer to bypass KatG activation. HPLC and EPR studies showed that the INH moiety can be oxidized by superoxide or peroxide yielding similar metabolites and isonicotinoyl radical only when associated to IQG-607, thereby supporting redox-mediated drug activation as a possible mechanism of action. However, IQG-607 was shown to inhibit the in vitro activity of both wild-type and INH-resistant mutant InhA enzymes in the absence of KatG activation. IQG-607 given by the oral route to M. tuberculosis-infected mice reduced lung lesions. Experiments using early and late controls of infection revealed a bactericidal activity for IQG-607. HPLC and voltammetric methods were developed to quantify IQG-607. Pharmacokinetic studies showed short half-life, high clearance, moderate volume of distribution, and low oral bioavailability, which was not altered by feeding. Safety and toxic effects of IQG-607 after acute and 90-day repeated oral administrations in both rats and minipigs showed occurrence of mild to moderate toxic events. Eight multidrug-resistant strains (MDR-TB) were resistant to IQG-607, suggesting an association between katG mutation and increasing MIC values. Whole genome sequencing of three spontaneous IQG-607-resistant strains harbored katG gene mutations. MIC measurements and macrophage infection experiments with a laboratorial strain showed that katG mutation is sufficient to confer resistance to IQG-607 and that the macrophage intracellular environment cannot trigger the self-activation mechanism. Reduced activity of IQG-607 against an M. tuberculosis strain overexpressing S94A InhA mutant protein suggested both the need for KatG activation and InhA as its target. Further efforts are suggested to be pursued toward attempting to translate IQG-607 into a chemotherapeutic agent to treat tuberculosis.

Template-induced self-activation route for nitrogen-doped hierarchically porous carbon spheres for electric double layer capacitors

The synthesis of highly nitrogen-doped hierarchical carbon spheres (NPCS) is reported. The NPCS with pomegranate-like nanostructure were prepared by the CaCO3 spheres template-induced self-activation route with dopamine as the carbon precursor. The template-induced self-activation mechanism was carefully investigated by controlling experiment conditions and thermogravimetric analyses combined with mass spectrometry. Under the optimized conditions, the as-prepared NPCS demonstrate large specific area (up to 1984 m2 g−1) and high level of nitrogen (N, 7.57%) doping. Electrochemical evaluations indicate that the energy density of NPCS-based EDLC devices can reach 6.2 and 14.4 Wh kg−1 in 6 M KOH electrolyte and aqueous concentrated electrolyte (5 M LiTFSI), respectively. This work provides a novel approach for preparing nanoporous carbon materials with hierarchical porosity and well-defined nanostructure.

In-situ activation of self-supported 3D hierarchically porous Ni3S2 films grown on nanoporous copper as excellent pH-universal electrocatalysts for hydrogen evolution reaction

The exploitation of low-cost, stable, and highly active electrocatalysts based on earth-abundant metals for hydrogen evolution reaction (HER) is crucial for developing renewable energy techniques. In this work, we report a facile synthesis strategy for in-situ fabrication of 3D hierarchically porous Ni3S2 films on a nanoporous copper substrate (Ni3S2@NPC) by unusual galvanic replacement reaction in the Ethaline-based deep eutectic solvent (DES) under a normal atmosphere. The self-supported nanoporous Ni3S2@NPC electrode is binder- free and exhibits good structural integrity with high conductivity. A mild evolution of bulk gas bubbles (H2-O2 gas mixture) is proved to drive an in-situ structure rearrangement process of the Ni3S2@NPC and results in substantial increases in the HER activity. The activated Ni3S2@NPC (a-Ni3S2@NPC) electrode can serve as a highly efficient and stable electrocatalyst for the HER in water over a wide pH range. Significantly, it displays high-performance HER catalytic activity in acidic media with robust durability over 111h and functions well under alkaline and neutral conditions. Such a superior catalytic performance of the a-Ni3S2@NPC is mainly due to the unique hierarchically nanoporous architectures and the synergetic effects in it caused by the restructuring NPC skeletons and active components. Our work offers a generic strategy for design and fabrication of many other self-supported transition metal sulfide and phosphide based HER electrocatalysts, and uncovers a new O2-induced electrochemical self-activation mechanism for improving the activity of catalysts.

A novel self-activation mechanism of Candida antarctica lipase B.

Candida antarctica lipase B (CalB), resembling many other lipases structure-wise, contains a flexible lid that undergoes a surprisingly large conformational change when catalyzing hydrophobic substrates (e.g. triglycerides). Despite extensive and important applications in industry, it is so far still elusive whether CalB can be activated on a hydrophobic surface, like other lipases. From large-scale all-atom molecular dynamics simulations, we discovered an open state that strikingly shows a much wider and more stable entrance to the catalytic site than the one suggested by previous crystal structures. Simulations demonstrate that in the newly found open state CalB possesses a "lid-holder" structure that intimately harbors the lid of CalB, i.e. a remarkable self-activation mechanism. To account for the unusual interfacial activation of CALB revealed in a recent experiment, we further introduce a simple model: the activation occurs only when the binding free energy between the lid and a hydrophobic surface is larger than a critical value, 4.0 kcal mol-1 that is the one between the lid and the "lid-holder". Our findings shed light on possible protein engineering of lipases to permit either self-activation with broadened catalytic targets (including water soluble ones) or surface activation with elevated activities.

A Self-Activated Mechanism for Nucleic Acid Polymerization Catalyzed by DNA/RNA Polymerases.

The enzymatic polymerization of DNA and RNA is the basis for genetic inheritance for all living organisms. It is catalyzed by the DNA/RNA polymerase (Pol) superfamily. Here, bioinformatics analysis reveals that the incoming nucleotide substrate always forms an H-bond between its 3'-OH and β-phosphate moieties upon formation of the Michaelis complex. This previously unrecognized H-bond implies a novel self-activated mechanism (SAM), which synergistically connects the in situ nucleophile formation with subsequent nucleotide addition and, importantly, nucleic acid translocation. Thus, SAM allows an elegant and efficient closed-loop sequence of chemical and physical steps for Pol catalysis. This is markedly different from previous mechanistic hypotheses. Our proposed mechanism is corroborated via ab initio QM/MM simulations on a specific Pol, the human DNA polymerase-η, an enzyme involved in repairing damaged DNA. The structural conservation of DNA and RNA Pols supports the possible extension of SAM to Pol enzymes from the three domains of life.

Enhanced brain release of erythropoietin, cytokines and NO during carotid clamping

Although effective and safe, carotid endarterectomy (CEA) implies a reduced blood flow to the brain and likely an ischemia/reperfusion event. The high rate of uneventful outcomes associated with CEA suggests the activation of brain endogenous protection mechanisms aimed at limiting the possible ischemia/reperfusion damage. This study aims at assessing whether CEA triggers protective mechanisms such as brain release of erythropoietin and nitric oxide. CEA was performed in 12 patients; blood samples were withdrawn simultaneously from the surgically exposed ipsilateral jugular and leg veins before, during (2 and 40 min) and after clamp removal (2 min). Plasma antioxidant capacity, carbonylated proteins, erythropoietin, nitrates and nitrites (NOx) were determined. No changes in intraoperative EEG, peripheral and transcranial blood oxygen saturation were detectable, and no patients showed any neurologic sign after the intervention. Antioxidant capacity and protein carbonylation in plasma were unaffected. Differently, erythropoietin, VEGF, TNF-α and NOx increased during clamping in the jugular blood (2 and 40 min), while no changes were observed in the peripheral circulation. These results show that blood erythropoietin, VEGF, TNF-α, and NOx increased in the brain during uncomplicated CEA. This may represent an endogenous self-activated neuroprotective mechanism aimed at the prevention of ischemia/reperfusion damage.

Self-Activation Mechanism Investigations on Large K2CO3-Doped Li4SiO4 Sorbent Particles

This work investigated the self-activation behavior of large K2CO3-doped Li4SiO4 sorbent particles. In this self-activation mechanism, the sorption ability increased as the number of cycles increased. After the sorption–desorption cycles occurred, the sorption ability of the K2CO3-doped Li4SiO4 sorbent was remarkably enhanced from approximately 2.0 mmol CO2/g sorbent to approximately 5.0 mmol CO2/g sorbent at 565 °C in 10 vol % CO2 atmosphere. The fresh and used sorbents were then characterized through N2 adsorption and SEM methods. Results showed that the average pore size increased from 7 to 32 nm and the surface microstructure changed from dense to porous, because the molten eutectic mixture formed by Li2CO3 and Li2SiO3 can facilitate CO2 diffusion. The formed CO2 diffusion channel can provide more CO2 accessibility; this channel can also reduce the CO2 diffusion resistance through the product layer. Therefore, the sorption ability of the sorbent is enhanced. Meanwhile, the effects of the self-activation temperature were also investigated and the results revealed that the optimal self-activation temperature is 615 °C. Furthermore, under critical conditions, the self-activated sorbent performed more efficiently than the fresh sample. At 450 °C under 10 vol % CO2 atmosphere, the sorption capacity of the self-activated sorbent was approximately 20 times higher than that of the fresh sample. Finally, a pore–core model was also proposed to illustrate the K2CO3-doped Li4SiO4 self-activation mechanism.

Self-activation of cellulose: A new preparation methodology for activated carbon electrodes in electrochemical capacitors

Current synthetic methods of biomass-derived activated carbon call for a costly chemical or physical activation process. Herein, we report a simple one-step annealing synthesis yielding a high surface area cellulose-derived activated carbon. We discover that simply varying the flow rate of Argon during pyrolysis enables ‘self-activation’ reactions that can tune the specific surface areas of the resulting carbon, ranging from 98m2/g to values as high as 2600m2/g. Furthermore, we, for the first time, observe a direct evolution of H2 from the pyrolysis, which gives strong evidence towards an in situ self-activation mechanism. Surprisingly, the obtained activated carbon is a crumbled graphene nanostructure composed of interconnected sheets, making it ideal for use in an electrochemical capacitor. The cellulose-derived nanoporous carbon exhibits a capacitance of 132Fg−1 at 1Ag−1, a performance comparable to the state-of-the-art activated carbons. This work presents a fundamentally new angle to look at the synthesis of activated carbon, and highlights the importance of a controlled inert gas flow rate during synthesis in general, as its contributions can have a very large impact on the final material properties.