Effect of Lanthanide Ions and Triazole Ligands on the Molecular Properties, Spectroscopy and Pharmacological Activity.

The effect of La, Ce, Pr and Nd ions on four Ln(ligand)3 complexes and at three DFT levels of calculation was analyzed. Four ligands were chosen, three of which were based on the 1,2,3-triazole ring. The DFT methods used were B3LYP, CAM-B3LYP and M06-2X. The relationships established were between the geometric parameters, atomic charges, HOMO-LUMO energies and other molecular properties. These comparisons and trends will facilitate the synthesis of new complexes by selecting the ligand and lanthanide ion best suited to the desired property of the complex. The experimental IR and Raman spectra of Ln(2b')3 complexes where Ln = La, Ce, Pr, Nd, Sm, Gd, Dy, Ho and Er ions have been recorded and compared to know the effect of the lanthanide ion on the complex. The hydration in these complexes was also analyzed. Additionally, the effect of the type of coordination center on the ability of an Ln(ligand)3 complex to participate in electron exchange and hydrogen transfer was investigated using two in vitro model systems-DPPH and ABTS.

Pretreatment methods in ion chromatography: A review.

As an advanced analytical technology, Ion Chromatography (IC) has been widely used in various fields. At present, it is faced with the challenges of sample complexity and instrument precision. It is necessary to select appropriate pretreatment methods to achieve sample preparation and protect the instruments. Therefore, this paper reviews several commonly used sample pretreatment technologies in IC, focusing on sample digestion and purification techniques. Additionally, we introduce some advanced IC technologies and automatic sample processing devices. We provide a comprehensive summary of the basic principles, primary applications and the advantages and disadvantages of each method. Pretreatment methods should be carefully selected and optimized on the specific characteristics of the sample and the ions to be measured, in order to achieve better analysis results.

An Insulin-Modified pH-Responsive Nanopipette Based on Ion Current Rectification.

The properties of nanopipettes largely rely on the materials introduced onto their inner walls, which allow for a vast extension of their sensing capabilities. The challenge of simultaneously enhancing the sensitivity and selectivity of nanopipettes for pH sensing remains, hindering their practical applications. Herein, we report insulin-modified nanopipettes with excellent pH response performances, which were prepared by introducing insulin onto their inner walls via a two-step reaction involving silanization and amidation. The pH response intensity based on ion current rectification was significantly enhanced by approximately 4.29 times when utilizing insulin-modified nanopipettes compared with bare ones, demonstrating a linear response within the pH range of 2.50 to 7.80. In addition, insulin-modified nanopipettes featured good reversibility and selectivity. The modification processes were monitored using the I-V curves, and the relevant mechanisms were discussed. The effects of solution pH and insulin concentration on the modification results were investigated to achieve optimal insulin introduction. This study showed that the pH response behavior of nanopipettes can be greatly improved by introducing versatile molecules onto the inner walls, thereby contributing to the development and utilization of pH-responsive nanopipettes.

A stepwise surface ionization method for ion mobility spectrometry.

RATIONALE: The detection of organic nitrogen compounds in exhaled breath is expected to provide an early warning of diseases such as kidney disease. Detecting these trace disease markers in exhaled breath with complex composition and high moisture content is a challenge. Surface ionization (SI) shows a highly selective ionization of organic nitrogen compounds, and it is a good candidate for breath analysis combined with ion mobility spectrometry (IMS). METHODS: A stepwise SI method of low-temperature adsorption/high-temperature ionization was proposed, and trimethylamine (TMA) was detected when combined with an ion mobility spectrometer. TMA at different concentrations and humidity levels and spiked in human breath was detected to evaluate the method's properties. RESULTS: TMA with concentrations from 2 to 200 ppb was detected. The peak intensity of the TMA characteristic ions was linearly related to the "e" exponent of the concentration with a curve fit of 0.996. A standard deviation of less than 0.306% was obtained with 10 replicate analyses of 10 ppb TMA. The signal intensity difference between dry and wet (relative humidity > 93%) TMA samples is only 2.7%, and the recovery rate of the sample was 106.819%. CONCLUSIONS: SI-IMS based on the stepwise SI method has the advantages of low ionization temperature, high detection sensitivity, strong resistance to humidity interference, and good repeatability. It is a promising method for detecting organic nitrogen compounds in exhaled breath.

Hyperconfined bio-inspired Polymers in Integrative Flow-Through Systems for Highly Selective Removal of Heavy Metal Ions.

Access to clean water, hygiene, and sanitation is becoming an increasingly pressing global demand, particularly owing to rapid population growth and urbanization. Phytoremediation utilizes a highly conserved phytochelatin in plants, which captures hazardous heavy metal ions from aquatic environments and sequesters them in vacuoles. Herein, we report the design of phytochelatin-inspired copolymers containing carboxylate and thiolate moieties. Titration calorimetry results indicate that the coexistence of both moieties is essential for the excellent Cd2+ ion-capturing capacity of the copolymers. The obtained dissociation constant, KD ~ 1 nM for Cd2+ ion, is four-to-five orders of magnitude higher than that for peptides mimicking the sequence of endogenous phytochelatin. Furthermore, infrared and nuclear magnetic resonance spectroscopy results unravel the mechanism underlying complex formation at the molecular level. The grafting of 0.1 g bio-inspired copolymers onto silica microparticles and cellulose membranes helps concentrate the copolymer-coated microparticles in ≈3 mL volume to remove Cd2+ ions from 0.3 L of water within 1 h to the drinking water level (<0.03 µM). The obtained results suggest that hyperconfinement of bio-inspired polymers in flow-through systems can be applied for the highly selective removal of harmful contaminants from the environmental water.

Review on Synthesis of 2-(2-Hydroxyaryl) Benzothiazoles (HBT) for Excited-State Intra-molecular Proton Transfer (ESIPT)-Based Detection of Ions and Biomolecules.

In this review, we present a systematic and comprehensive summary of the recent developments in the synthetic strategies of 2-(2-hydroxyarylsubstituted)-benzothiazole (HBT) framework along with incorporation of various substituents on phenolic and benzothiazole rings which affect the emission process. The literature, spanning the years 2015-2024, on excited-state intramolecular proton transfer (ESIPT)-based studies of HBT derivatives comprising the effects of solvent polarity, substituents, and extended conjugation on fluorophores has been searched. ESIPT, intramolecular charge transfer, and aggregation-induced emissions enable these fluorescent probes to specifically interact with analytes, thereby altering their luminescence characteristics to achieve analyte detection. These fluorescent probes exhibit large Stokes shifts, high quantum yields, and excellent color transitions. Finally, the applications of HBTs as ESIPT-based fluorescent probes for the detection of cations, anions, and biomolecules have been summarized. We anticipate that this review will provide a comprehensive overview of the current state of research in this field and encourage researchers to develop novel ESIPT-based fluorophores with new applications.

19F-NMR Probing of Ion-Induced Conformational Changes in Detergent-Solubilized and Nanodisc-Reconstituted NCX_Mj.

Consecutive interactions of 3Na+ or 1Ca2+ with the Na+/Ca2+ exchanger (NCX) result in an alternative exposure (access) of the cytosolic and extracellular vestibules to opposite sides of the membrane, where ion-induced transitions between the outward-facing (OF) and inward-facing (IF) conformational states drive a transport cycle. Here, we investigate sub-state populations of apo and ion-bound species in the OF and IF states by analyzing detergent-solubilized and nanodisc-reconstituted preparations of NCX_Mj with 19F-NMR. The 19F probe was covalently attached to the cysteine residues at entry locations of the cytosolic and extracellular vestibules. Multiple sub-states of apo and ion-bound species were observed in nanodisc-reconstituted (but not in detergent-solubilized) NCX_Mj, meaning that the lipid-membrane environment preconditions multiple sub-state populations toward the OF/IF swapping. Most importantly, ion-induced sub-state redistributions occur within each major (OF or IF) state, where sub-state interconversions may precondition the OF/IF swapping. In contrast with large changes in population redistributions, the sum of sub-state populations within each inherent state (OF or IF) remains nearly unchanged upon ion addition. The present findings allow the further elucidation of structure-dynamic modules underlying ion-induced conformational changes that determine a functional asymmetry of ion access/translocation at opposite sides of the membrane and ion transport rates concurring physiological demands.

Conformation-Selective Ultraviolet-Ultraviolet Hole Burning Spectra of Ubiquitin Ions in a Cryogenic Ion Trap.

Understanding the structural variations of conformational isomers in proteins is crucial for elucidating protein folding mechanisms. Here, we present a novel method for obtaining conformation-selective ultraviolet (UV)-UV hole burning (HB) spectra of ubiquitin ions ((Ubi+zH)+z, z = 7-10) produced via electrospray ionization. Our approach involves binding multiple N2 molecules to ubiquitin ions ((Ubi+zH)+z(N2)m, m = 1-55) within a cryogenic ion trap. Upon exposure to UV irradiation, efficient fragmentation of (Ubi+zH)+z(N2)m occurs, primarily yielding bare (Ubi+zH)+z ions as fragments. The significant mass difference between the parent and fragment ions facilitates the acquisition of UV-UV HB spectra, which reveal the presence of at least two distinct conformers. Molecular dynamics simulations suggest that these conformers correspond to A-state structures, differing only in the interactions of a tyrosine residue with neighboring residues. Our findings underscore UV-UV HB spectroscopy of protein ions as a powerful tool for exploring diverse protein isomers.

Wound Healing Promotion via Release of Therapeutic Metallic Ions from Phosphate Glass Fibers: An In Vitro and Ex Vivo Study.

Biomaterials capable of promoting wound healing and preventing infections remain in great demand to address the global unmet need for the treatment of chronic wounds. Phosphate-based glasses (PG) have shown potential as bioresorbable materials capable of inducing tissue regeneration, while being replaced by regenerated tissue and releasing therapeutic species. In this work, phosphate-glass-based fibers (PGF) in the system P2O5-CaO-Na2O added with 1, 2, 4, 6, and 10 mol % of the therapeutic metallic ions (TMI) Ag+, Zn2+, and Fe3+ were manufactured via electrospinning of coacervate gels. Coacervation is a sustainable, cost-effective, water-based method to produce PG. All TMI are effective in promoting wound closure (re-epithelialization) in living human skin ex vivo, where the best-performing system is PGF containing Ag+. In particular, PGF with ≥4 mol % of Ag+ is capable of promoting 84% wound closure over 48 h. These results are confirmed by scratch test migration assays, with the PGF-Ag systems containing ≥6 mol % of Ag+, demonstrating significant wound closure enhancement (up to 72%) after 24 h. The PGF-Ag systems are also the most effective in terms of antibacterial activity against both the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli. PGF doped with Zn2+ shows antibacterial activity only against S. aureus in the systems containing Zn2+ ≥ 10 mol %. In addition, PGF doped with Fe3+ rapidly accelerates ex vivo healing in patient chronic wound skin (>30% in 48 h), demonstrating the utility of doped PGF as a potential therapeutic strategy to treat chronic wounds.

Silver/gold nanoalloy implant coatings with antibiofilm activity via pH-triggered silver ion release.

Implant infections are a major challenge for the healthcare system. Biofilm formation and increasing antibiotic resistance of common bacteria cause implant infections, leading to an urgent need for alternative antibacterial agents. In this study, the antibiofilm behaviour of a coating consisting of a silver (Ag)/gold (Au) nanoalloy is investigated. This alloy is crucial to reduce uncontrolled potentially toxic Ag+ ion release. In neutral pH environments this release is minimal, but the Ag+ ion release increases in acidic microenvironments caused by bacterial biofilms. We perform a detailed physicochemical characterization of the nanoalloys and compare their Ag+ ion release with that of pure Ag nanoparticles. Despite a lower released Ag+ ion concentration at pH 7.4, the antibiofilm activity against Escherichia coli (a bacterium known to produce acidic pH environments) is comparable to a pure nanosilver sample with a similar Ag-content. Finally, biocompatibility studies with mouse pre-osteoblasts reveal a decreased cytotoxicity for the alloy coatings and nanoparticles.

Nobody's Perfect: Choice of the Buffer and the Rate of Cu2+ Ion-Peptide Interaction.

The choice of correct pH buffer is crucial in chemical studies modeling biological processes involving Cu2+ ions. Popular buffers for physiological pH are known to form Cu(II) complexes, but their impact on kinetics of Cu(II) complexation has not been considered. We performed a stopped-flow kinetic study of Cu2+ ion interactions with four popular buffers (phosphate, Tris, HEPES, and MOPS) and two buffers considered as nonbinding (MES and PIPPS). Next, we studied their effects on the rate of Cu2+ reaction with Gly-Gly-His (GGH), a tripeptide modeling physiological Cu(II) sites, which we studied previously at conditions presumably excluding the buffer interference [Kotuniak, R.; Angew. Chem., Int. Ed. 2020, 59, 11234-11239]. We observed that (i) all tested pH 7.4 buffers formed Cu(II) complexes within the stopped-flow instrument dead time; (ii) Cu(II)-peptide complexes were formed via ternary complexes with the buffers; (iii) nevertheless, Good buffers affected the observed rate of Cu(II)-GGH complex formation only slightly; (iv) Tris was a competitive inhibitor of Cu(II)-GGH complexation; while (v) phosphate was a reaction catalyst. This is particularly important as phosphate is a biological buffer.

Experimentally Determined Diagnostic Ions for Identification of Peptide Glycotopes.

The analysis of the structures of glycans present on glycoproteins is an essential component for determining glycoprotein function; however, detailed glycan structural assignment on glycopeptides from proteomics mass spectrometric data remains challenging. Glycoproteomic analysis by mass spectrometry currently can provide significant, yet incomplete, information about the glycans present, including the glycan monosaccharide composition and in some circumstances the site(s) of glycosylation. Advancements in mass spectrometric resolution, using high-mass accuracy instrumentation and tailored MS/MS fragmentation parameters, coupled with a dedicated definition of diagnostic fragmentation ions have enabled the determination of some glycan structural features, or glycotopes, expressed on glycopeptides. Here we present a collation of diagnostic glycan fragments produced by traditional positive-ion-mode reversed-phase LC-ESI MS/MS proteomic workflows and describe the specific fragmentation energy settings required to identify specific glycotopes presented on N- or O-linked glycopeptides in a typical proteomics MS/MS experiment.

Removal of trivalent chromium ions in model contaminated groundwater using hexagonal boron nitride as an adsorbent.

The feasibility of using hexagonal boron nitride (h-BN) to treat heavy metal Cr(III) from model contaminated groundwater was evaluated in this study by adsorption experiments and characterizations. To the best of our knowledge, this study is the first attempt to conduct the adsorption of Cr(III) by h-BN under various experimental conditions such as exposure time, ratio of adsorbates and adsorbents, solution pH, background ions with different ionic strength, and the presence of humic acids (HA) in model contaminated groundwater. The optimized h-BN showed excellent maximum adsorption capacity (i.e., 177 mg ∙ g-1) when the concentrations of Cr(III) and h-BN were 10 and 10 mg ∙ L-1, respectively. Subsequently, we confirmed there was a negligible change in the adsorption performance of Cr(III) by h-BN in the presence of co-ions (i.e., K and Mg) in concentrations in a range from 50 to 1000 mg ∙ L-1. Furthermore, the adsorption performance of Cr(III) gradually improved with HA concentrations from 2.5 to 25 mg ∙ L-1. Interestingly, the maximum adsorption performance of Cr(III) by both HA and h-BN increased until 500 mg ∙ g-1 in the presence of 25 mg ∙ L-1 HA. The adsorption mechanism was clarified by Fourier-transform infrared spectroscopy and X-ray photoelectron spectroscopy. Additionally, we successfully confirmed that h-BN could be reused until five cycles. On the basis of the adsorption performance results and characterizations, h-BN can be utilized as an efficient and practical adsorbent to treat Cr(III) in groundwater treatment.

Detecting Bound Ions in Ion Channels by Solid-State NMR Experiments on 15N-Labelled Ammonium Ions.

Solid-state NMR allows for the study of membrane proteins under physiological conditions. Here we describe a method for detection of bound ions in the selectivity filter of ion channels using solid-state NMR. This method employs standard 1H-detected solid-state NMR setup and experiment types, which is enabled by using 15N-labelled ammonium ions to mimic potassium ions.

Salt ions improve soybean protein isolate/curdlan complex fat substitutes: Effect of molecular interactions on freeze-thaw stability.

Although emulsion gels show significant potential as fat substitutes, they are vulnerable to degreasing, delamination, and other undesirable processes during freezing, storage, and thawing, leading to commercial value loss in terms of juiciness, flavor, and texture. This study investigated the gel strength and freeze-thaw stability of soybean protein isolate (SPI)/curdlan (CL) composite emulsion gels after adding sodium chloride (NaCl). Analysis revealed that adding low salt ion concentrations promoted the hardness and water-holding capacity (WHC) of fat substitutes, while high levels displayed an inhibitory effect. With 40 mM NaCl as the optimum concentration, the hardness increased from 259.33 g (0 mM) to 418.67 g, the WHC increased from 90.59 % to 93.18 %, exhibiting good freeze-thaw stability. Confocal laser scanning microscopy (CLSM) and particle size distribution were used to examine the impact of salt ion concentrations on protein particle aggregation and the damaging effect of freezing and thawing on the proteoglycan complex network structure. Fourier-transform infrared spectroscopy (FTIR) and protein solubility evaluation indicated that the composite gel network structure consisted of covalent contacts between the proteoglycan molecules and hydrogen bonds, playing a predominant role in non-covalent interaction. This study showed that the salt ion concentration in the emulsion gel affected its molecular interactions.

Antibacterial Action of Zn2+ Ions Driven by the In Vivo Formed ZnO Nanoparticles.

Antibacterial formulations based on zinc oxide nanoparticles (ZnO NPs) are widely used for antibiotic replacement in veterinary medicine and animal nutrition. However, the undesired environmental impact of ZnO NPs triggers a search for alternative, environmentally safer solutions. Here, we show that Zn2+ in its ionic form is a more eco-friendly antibacterial, and its biocidal action rivals that of ZnO NPs (<100 nm size), with a minimal biocidal concentration being 41(82) µg mL-1 vs 5 µg mL-1 of ZnO NPs, as determined for 103(106) CFU mL-1 E. coli. We demonstrate that the biocidal activity of Zn2+ ions is primarily associated with their uptake by E. coli and spontaneous in vivo transformation into insoluble ZnO nanocomposites at an internal bacterial pH of 7.7. Formed in vivo nanocomposite then damages E. coli membrane and intracellular components from the inside, by forming insoluble biocomposites, whose formation can also trigger ZnO characteristic reactions damaging the cells (e.g., by generation of high-potential reactive oxygen species). Our study defines a special route in which Zn2+ metal ions induce the death of bacterial cells, which might be common to other metal ions capable of forming semiconductor oxides and insoluble hydroxides at a slightly alkaline intracellular pH of some bacteria.

Elucidating the Gas-Phase Behavior of Nitazene Analog Protomers Using Structures for Lossless Ion Manipulations Ion Mobility-Orbitrap Mass Spectrometry.

2-Benzylbenzimidazoles, or "nitazenes", are a class of novel synthetic opioids (NSOs) that are increasingly being detected alongside fentanyl analogs and other opioids in drug overdose cases. Nitazenes can be 20× more potent than fentanyl but are not routinely tested for during postmortem or clinical toxicology drug screens; thus, their prevalence in drug overdose cases may be under-reported. Traditional analytical workflows utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) often require additional confirmation with authentic reference standards to identify a novel nitazene. However, additional analytical measurements with ion mobility spectrometry (IMS) may provide a path toward reference-free identification, which would greatly accelerate NSO identification rates in toxicology laboratories. Presented here are the first IMS and collision cross section (CCS) measurements on a set of fourteen nitazene analogs using a structures for lossless ion manipulations (SLIM)-orbitrap MS. All nitazenes exhibited two high intensity baseline-separated IMS distributions, which fentanyls and other drug and druglike compounds also exhibit. Incorporating water into the electrospray ionization (ESI) solution caused the intensities of the higher mobility IMS distributions to increase and the intensities of the lower mobility IMS distributions to decrease. Nitazenes lacking a nitro group at the R1 position exhibited the greatest shifts in signal intensities due to water. Furthermore, IMS-MS/MS experiments showed that the higher mobility IMS distributions of all nitazenes possessing a triethylamine group produced fragment ions with m/z 72, 100, and other low intensity fragments while the lower mobility IMS distributions only produced fragment ions with m/z 72 and 100. The IMS, solvent, and fragmentation studies provide experimental evidence that nitazenes potentially exhibit three gas-phase protomers. The cyclic IMS capability of SLIM was also employed to partially resolve four sets of structurally similar nitazene isomers (e.g., protonitazene/isotonitazene, butonitazene/isobutonitazene/secbutonitazene), showcasing the potential of using high-resolution IMS separations in MS-based workflows for reference-free identification of emerging nitazenes and other NSOs.

Design, preparation, and performance of different adsorbents based on carboxymethyl chitosan/sodium alginate hydrogel beads for selective adsorption of Cadmium (II) and Chromium (III) metal ions.

Developing cost-effective and efficient adsorbents for heavy metals in multicomponent systems is a challenge that needs to be resolved to meet the challenges of wastewater treatment technology. Two adsorbents were synthesized, characterized, and investigated for the removal of Cd2+ and Cr3+ as model heavy metals in their single and binary solutions. The first adsorbent (ACZ) was a nanocomposite formed of O-Carboxymethyl chitosan, sodium alginate, and zeolite. While, the other (ACL) contained ZnFe layered double hydroxides instead of the zeolite phase. Adsorbents were characterized using XRD, FTIR, SEM, and swelling degree analysis. For single heavy metal adsorption isotherms, data for both adsorbents was best fitted and indicated a multilayer adsorption nature. For binary adsorption, Langmuir model with interacting parameters showed the best results compared to other models for both pollutants. For single system, Avrami model was found to be the best model representing the adsorption kinetics data, which indicates that the mechanism of adsorption follows multiple kinetic orders that may change during duration of adsorption process. Numerous interaction mechanisms can occur between the heavy metals and functional groups in the synthesized hydrogels such as NH2, COOH, and OH groups leading to efficient adsorption of metal ions.

Selective uranyl ion-imprinting with clickable amidoxime-functionalized pullulan.

Manufacturing a highly effective sorbent for removing UO22+ ions from aqueous effluents is vital for safeguarding the environment and recovering valuable resources. This research presents an innovative strategy employing adsorbents derived from pullulan, specifically tailored with furfuryl-amidoxime (FAO), to improve their affinity for UO22+ ions. The formation of a UO22+ ion-imprinted sorbent (U-II-P) was achieved by crosslinking the UO22+/FAO-modified pullulan (FAO-P) complex with bis(maleimido)ethane (BME) via click Diels-Alder (DA) cyclization, enhancing its attraction and specificity for UO22+ ions. Detailed characterization of the synthesis was performed using NMR and FTIR spectroscopy, and the sorbent's external textures were analyzed using scanning electron microscopy (SEM). The U-II-P sorbent showcased outstanding preference for UO22+ over other metallic ions, with the most efficient adsorption occurring at pH 5. It exhibited a significant adsorption capacity of 262 mg/g, closely aligning with the predictions of the Langmuir adsorption model and obeying pseudo-second-order kinetic behavior. This investigation underlines the effectiveness of FAO-P as a specialized solution for UO22+ ion extraction from wastewater, positioning it as a viable option for the remediation of heavy metals.

Designable immobilization of D-allulose 3-epimerase on bimetallic organic frameworks based on metal ion compatibility for enhanced D-allulose production.

D-allulose, a low-calorie rare sugar catalyzed by D-allulose 3-epimerase (DAE), is highly sought after for its potential health benefits. However, poor reusability and stability of DAE limited its popularization in industrial applications. Although metal-organic frameworks (MOFs) offer a promising enzyme platform for enzyme immobilization, developing customized strategies for MOF immobilization of enzymes remains challenging. In this study, we introduce a designable strategy involving the construction of bimetal-organic frameworks (ZnCo-MOF) based on metal ions compatibility. The DAE@MOFs materials were prepared and characterized, and the immobilization of DAE and the enzymatic characteristics of the MOF-immobilized DAE were subsequently evaluated. Remarkably, DAE@ZnCo-MOF exhibited superior recyclability which could maintain 95 % relative activity after 8 consecutive cycles. The storage stability is significantly improved compared to the free form, with a relative activity of 116 % remaining after 30 days. Molecular docking was also employed to investigate the interaction between DAE and the components of MOFs synthesis. The results demonstrate that the DAE@ZnCo-MOF exhibited enhanced catalytic efficiency and increased stability. This study introduces a viable and adaptable MOF-based immobilization strategy for enzymes, which holds the potential to expand the implementation of enzyme biocatalysts in a multitude of disciplines.