Quantification of GTPase Cycling Rates of GTPases and GTPase:Effector Mixtures Using GTPase Glo Assays.

In different cellular activities such as signal transduction, cell division, and intracellular transportation, small guanosine triphosphatases (GTPases) take on a vital role. Their function involves hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP). In this article, we explain the application of a commercially available GTPase assay-the GTPase Glo assay by Promega-for investigation of GTPase-effector interactions. We provide experimental protocols together with an analysis model and software to obtain GTPase cycling rates of GTPases and GTPase:effector mixtures. GTPase cycling rates refer to the rates by which a GTPase completes an entire GTPase cycle. These rates enable quantification of the strength of GTPase effectors in a concentration-dependent fashion, as well as quantification of the combined effect of two effectors, independent of which GTPase cycle step they are affecting. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Conducting GTPase Glo assays Support Protocol 1: Analyzing GTPase assays to correlate luminescence with remaining GTP Support Protocol 2: Fitting GTPase assay data to obtain GTPase cycling rates.

Preparation, assay, and application of 4-fluorothreonine transaldolase from Streptomyces sp. MA37 for ß-hydroxyl amino acid derivatives.

ß-Hydroxy-α-amino acids (ßHAAs) are an essential class of building blocks of therapeutically important compounds and complex natural products. They contain two chiral centers at Cα and Cß positions, resulting in four possible diastereoisomers. Many innovative asymmetric syntheses have been developed to access structurally diverse ßHAAs. The main challenge, however, is the control of the relative and absolute stereochemistry of the asymmetric carbons in a sustainable way. In this respect, there has been considerable attention focused on the chemoenzymatic synthesis of ßHAAs via a one-step process. Nature has evolved different enzymatic routes to produce these valuable ßHAAs. Among these naturally occurring transformations, L-threonine transaldolases present potential biocatalysts to generate ßHAAs in situ. 4-Fluorothreonine transaldolase from Streptomyces sp. MA37 (FTaseMA) catalyzes the cross-over transaldolation reaction between L-Thr and fluoroacetaldehyde to give 4-fluorothreonine and acetaldehyde (Ad). It has been demonstrated that FTaseMA displays considerable substrate plasticity toward structurally diverse aldehyde acceptors, leading to the production of various ßHAAs. In this chapter, we describe methods for the preparation of FTaseMA, and the chemoenzymatic synthesis of ßHAAs from various aldehydes and L-Thr using FTaseMA.

Microwell fluoride assay screening for enzymatic defluorination.

There is intense interest in removing fluorinated compounds from the environment, environments are most efficiently remediated by microbial enzymes, and defluorinating enzymes are readily monitored by fluoride determination. Fluorine is the most electronegative element. Consequently, all mechanisms of enzymatic C-F bond cleavage produce fluoride anion, F-. Therefore, methods for the determination of fluoride are critical for C-F enzymology and apply to any fluorinated organic compounds, including PFAS, or per- and polyfluorinated alkyl substances. The biodegradation of most PFAS chemicals is rare or unknown. Accordingly, identifying new enzymes, or re-engineering the known defluorinases, will require rapid and sensitive methods for measuring fluoride in aqueous media. Most studies currently use ion chromatography or fluoride specific electrodes which are relatively sensitive but low throughput. The methods here describe refashioning a drinking water test to efficiently determine fluoride in enzyme and cell culture reaction mixtures. The method is based on lanthanum alizarin complexone binding of fluoride. Reworking the method to a microtiter well plate format allows detection of as little as 4 nmol of fluoride in 200 µL of assay buffer. The method is amenable to color imaging, spectrophotometric plate reading and automated liquid handling to expedite assays with thousands of enzymes and/or substrates for discovering and improving enzymatic defluorination.

Fluorescent metal nanoclusters: From luminescence mechanism to applications in enzyme activity assays.

Metal nanoclusters (MNCs) have outstanding fluorescence property and biocompatibility, which show widespread applications in biological analysis. Particularly, evaluation of enzyme activity with the fluorescent MNCs has been developed rapidly within the past several years. In this review, we first introduced the fluorescent mechanism of mono- and bi-metallic nanoclusters, respectively, whose interesting luminescence properties are mainly resulted from electron transfer between the lowest unoccupied molecular orbital (LUMO) and highest occupied molecular orbital (HOMO) energy levels. Meanwhile, the charge migration within the structure occurs through ligand-metal charge transfer (LMCT) or ligand-metal-metal charge transfer (LMMCT). On such foundation, diverse enzyme activities were rigorously evaluated, including three transferases and nine hydrolases, in turn harvesting rapid research progresses within past 5 years. Finally, we summarized the design strategies for evaluating enzyme activity with the MNCs, presented the major issues and challenges remained in the relevant research, coupled by showing some improvement measures. This review will attract researchers dedicated to the studies of the MNCs and provide some constructive insights for their further applications in enzyme analysis.

Application of Fenton chemistry in electrochemical determination of pyrophosphatase activity and fluoride.

Fenton chemistry has aroused widespread concern due to its application in the green oxidation and mineralization of organic wastes. Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of pyrophosphate ions (PPi) and provides a thermodynamic driving force for many biosynthetic reactions. Fluoride (F-) is widely applied to fight against tooth decay and reduce cavities. The electrochemical determination of PPase activity and F- was realized based on Fenton chemistry in this work. Glassy carbon electrode modified with poly (azure A) and acetylene black (GCE/PAA-AB) was fabricated. Hydroxyl radicals (∙OH) that were generated from a Cu2+-catalyzed Fenton-type reaction could oxidize PAA in the near-neutral medium, leading to a great increase of the cathodic peak current (Ipc). A coordination reaction between PPi and Cu2+ exerted a negative effect on Fenton reaction and hindered the Ipc enhancement. Cu2+-PPi complex was decomposed due to the hydrolysis of PPi induced by PPase, which caused the reappearance of the notably increased current response. F- could effectively inhibit PPase activity. As a result, the stable Cu2+-PPi complex remained and the high Ipc suffered from the decline again. The Ipc difference was used for the highly sensitive determination of PPase activity in the content range of 0.001-20 mU mL-1 with a detection of limit (LOD) at 0.6 µU mL-1 and that of F- in the concentration range of 0.01-100 µM with a LOD at 7 nM. The proposed PPase and F- sensor displayed a good selectivity, stability and reproducibility, and a high accuracy.

Prolidase activity assays. A survey of the reported literature methodologies.

Prolidase (EC.3.4.13.9) is a dipeptidase known nowadays to play a pivotal role in several physiological and pathological processes. More in particular, this enzyme is involved in the cleavage of proline- and hydroxyproline-containing dipeptides (imidodipeptides), thus finely regulating the homeostasis of free proline and hydroxyproline. Abnormally high or low levels of prolidase have been found in numerous acute and chronic syndromes affecting humans (chronic liver fibrosis, viral and acute hepatitis, cancer, neurological disorders, inflammation, skin diseases, intellectual disability, respiratory infection, and others) for which the content of proline is well recognized as a clinical marker. As a consequence, the accurate analytical determination of prolidase activity is of greatly significant importance in clinical diagnosis and therapy. Apart from the Chinard's assay, some other more sensitive and well validated methodologies have been published. These include colorimetric and spectrophotometric determinations of free proline produced by enzymatic reactions, capillary electrophoresis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, electrochemoluminescence, thin layer chromatography, and HPLC. The aim of this comprehensive review is to make a detailed survey of the in so far reported analytical techniques, highlighting their general features, as well as their advantages and possible drawbacks, providing in the meantime suggestions to stimulate further research in this intriguing field.

Detection of terminal deoxynucleotidyl transferase activity based on self-mediated nucleic acid elongation and elemental labeling inductively coupled plasma-mass spectrometry.

Terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase, is recognized as a promising biomarker for acute leukemia. Herein, taking the advantage of the self-mediated strand elongation property of TdT, a simple and sensitive method for TdT activity assay was developed based on gold nanoparticles (AuNPs) labeling inductively coupled plasma mass spectrometry (ICP-MS). In the presence of TdT, the primer DNA on magnetic beads is elongated with an adenine-rich single stranded long chain that can label poly-thymine modified AuNPs. After acid elution, the labeled AuNPs were detected by ICP-MS, and the signal intensity of 197Au reflected the TdT activity. Under the optimal conditions, the limit of detection for TdT activity is down to 0.054 U mL-1, along with good selectivity and strong tolerance to other interfering proteins. Furthermore, it achieves a straightforward and accurate detection of TdT activity in acute lymphoblastic leukemia cells without sample pre-processing and tool enzyme addition. Therefore, the proposed method shows great promise as a valuable tool for TdT-related biological research and leukemia therapeutics.

Streamlining assays of glycosyltransferases activity using in vitro GT-array (i-GT-ray) platform: Application to family GT37 fucosyltransferases.

Numerous putative glycosyltransferases (GTs) have been identified using bioinformatic approaches. However, demonstrating the activity of these GTs remains a challenge. Here, we describe the development of a rapid in vitro GT-array screening platform for activity of GTs. GT-arrays are generated by cell-free in vitro protein synthesis and binding using microplates precoated with a N-terminal Halo- or a C-terminal GST-tagged GT-encoding plasmid DNA and a capture antibody. These arrays are then used for screening of transferase activities and the reactions are monitored by a luminescence GLO assay. The products formed by these reactions can be analyzed directly from the microplates by mass spectrometry. Using this platform, a total of 280 assays were performed to screen 22 putative fucosyltransferases (FUTs) from family GT37 (seven from Arabidopsis and 15 from rice) for activity toward five acceptors: non-fucosylated tamarind xyloglucan (TXyG), arabinotriose (Ara3), non-fucosylated rhamnogalacturonan I (RG-I), and RG-II from the mur1-1 Arabidopsis mutant, and the celery RG-II monomer lacking Arap and MeFuc of chain B and l-Gal of chain A. Our screen showed that AtFUT2, AtFUT5, and AtFUT10 have activity toward RG-I, while AtFUT8 was active on RG-II. Five rice OsFUTs have XyG-FUT activity and four rice OsFUTs have activity toward Ara3. None of the putative OsFUTs were active on the RG-I and RG-II. However, promiscuity toward acceptors was observed for several FUTs. These findings extend our knowledge of cell wall polysaccharide fucosylation in plants. We believe that in vitro GT-array platform provides a valuable tool for cell wall biochemistry and other research fields.

The biochemically defined super relaxed state of myosin-A paradox.

The biochemical SRX (super-relaxed) state of myosin has been defined as a low ATPase activity state. This state can conserve energy when the myosin is not recruited for muscle contraction. The SRX state has been correlated with a structurally defined ordered (versus disordered) state of muscle thick filaments. The two states may be linked via a common interacting head motif (IHM) where the two heads of heavy meromyosin (HMM), or myosin, fold back onto each other and form additional contacts with S2 and the thick filament. Experimental observations of the SRX, IHM, and the ordered form of thick filaments, however, do not always agree, and result in a series of unresolved paradoxes. To address these paradoxes, we have reexamined the biochemical measurements of the SRX state for porcine cardiac HMM. In our hands, the commonly employed mantATP displacement assay was unable to quantify the population of the SRX state with all data fitting very well by a single exponential. We further show that mavacamten inhibits the basal ATPases of both porcine ventricle HMM and S1 (Ki, 0.32 and 1.76 µM respectively) while dATP activates HMM cooperatively without any evidence of an SRX state. A combination of our experimental observations and theories suggests that the displacement of mantATP in purified proteins is not a reliable assay to quantify the SRX population. This means that while the structurally defined IHM and ordered thick filaments clearly exist, great care must be employed when using the mantATP displacement assay.

Sensitive Silver-Enhanced Microplate Apta-Enzyme Assay of Sb3+ Ions in Drinking and Natural Waters.

The toxic effects of antimony pose risks to human health. Therefore, simple analytical techniques for its widescale monitoring in water sources are in demand. In this study, a sensitive microplate apta-enzyme assay for Sb3+ detection was developed. The biotinylated aptamer A10 was hybridized with its complementary biotinylated oligonucleotide T10 and then immobilized on the surface of polysterene microplate wells. Streptavidin labeled with horseradish peroxidase (HRP) bound to the biotin of a complementary complex and transformed the 3,3',5,5'-tetramethylbenzidine substrate, generating an optical signal. Sb3+ presenting in the sample bounded to an A10 aptamer, thus releasing T10, preventing streptavidin-HRP binding and, as a result, reducing the optical signal. This effect allowed for the detection of Sb3+ with a working range from 0.09 to 2.3 µg/mL and detection limit of 42 ng/mL. It was established that the presence of Ag+ at the stage of A10/T10 complex formation promoted dehybridization of the aptamer A10 and the formation of the A10/Sb3+ complex. The working range of the Ag+-enhanced microplate apta-enzyme assay for Sb3+ was determined to be 8-135 ng/mL, with a detection limit of 1.9 ng/mL. The proposed enhanced approach demonstrated excellent selectivity against other cations/anions, and its practical applicability was confirmed through an analysis of drinking and spring water samples with recoveries of Sb3+ in the range of 109.0-126.2% and 99.6-106.1%, respectively.

In Vitro Assay to Measure APE1 Enzymatic Activity on Ribose Monophosphate Abasic Site.

APE1 (apurinic/apyrimidinic endodeoxyribonuclease 1) is a central enzyme of the base excision repair (BER) pathway playing a pivotal role in protecting mammalian cells against genotoxins and in safeguarding genome stability. Recently, we demonstrated the APE1 ability to process abasic ribonucleotides embedded in DNA. Here, we provide a pipeline of protocols to quantify endodeoxyribonuclease activity by APE1 on these substrates, by using recombinant protein and whole-cell extracts. The repair capacity is measured by using fluorescent oligonucleotide substrates, which are then separated by polyacrylamide gel electrophoresis and detected by imaging scanning. The specificity of APE1 action is demonstrated using specific APE1 enzymatic inhibitors.

Digital Cascade Assays for ADP- or ATP-Producing Enzymes Using a Femtoliter Reactor Array Device.

Digital enzyme assays are emerging biosensing methods for highly sensitive quantitative analysis of biomolecules with single-molecule detection sensitivity. However, current digital enzyme assays require a fluorogenic substrate for detection, which limits the applicability of this method to certain enzymes. ATPases and kinases are representative enzymes for which fluorogenic substrates are not available; however, these enzymes form large domains and play a central role in biology. In this study, we implemented a fluorogenic cascade reaction in a femtoliter reactor array device to develop a digital bioassay platform for ATPases and kinases. The digital cascade assay enabled quantitative measurement of the single-molecule activity of F1-ATPase, the catalytic portion of ATP synthase. We also demonstrated a digital assay for human choline kinase α. Furthermore, we developed a digital cascade assay for ATP-synthesizing enzymes and demonstrated a digital assay for pyruvate kinase. These results show the high versatility of this assay platform. Thus, the digital cascade assay has great potential for the highly sensitive detection and accurate characterization of various ADP- and ATP-producing enzymes, such as kinases, which may serve as disease biomarkers.

An Enzymatic Activity Assay for Heparanase That Is Useful for Evaluating Clinically Relevant Inhibitors and Studying Kinetics.

The enzyme heparanase cleaves heparan sulfate and is involved in a range of human diseases including cancer, inflammation, diabetes, and viral infection. There is a need for a simple and reliable enzymatic assay to allow for the screening of compounds to find inhibitors of heparanase. We have developed an assay that uses the heparinoid fondaparinux as enzyme substrate and detects one of the products of catalysis, which contains a newly formed reducing terminus, with the tetrazolium salt WST-1. Due to the homogenous substrate and single point of cleavage therein, this assay allows for more systematic kinetic analysis of heparanase inhibitors. Here, we provide a detailed method for conducting this assay and also provide information to assist researchers in evaluating whether the assay is performing properly in their laboratories.

Mass spectrometry imaging-based assays for aminotransferase activity reveal a broad substrate spectrum for a previously uncharacterized enzyme.

Aminotransferases (ATs) catalyze pyridoxal 5'-phosphate-dependent transamination reactions between amino donor and keto acceptor substrates and play central roles in nitrogen metabolism of all organisms. ATs are involved in the biosynthesis and degradation of both proteinogenic and nonproteinogenic amino acids and also carry out a wide variety of functions in photorespiration, detoxification, and secondary metabolism. Despite the importance of ATs, their functionality is poorly understood as only a small fraction of putative ATs, predicted from DNA sequences, are associated with experimental data. Even for characterized ATs, the full spectrum of substrate specificity, among many potential substrates, has not been explored in most cases. This is largely due to the lack of suitable high-throughput assays that can screen for AT activity and specificity at scale. Here we present a new high-throughput platform for screening AT activity using bioconjugate chemistry and mass spectrometry imaging-based analysis. Detection of AT reaction products is achieved by forming an oxime linkage between the ketone groups of transaminated amino donors and a probe molecule that facilitates mass spectrometry-based analysis using nanostructure-initiator mass spectrometry or MALDI-mass spectrometry. As a proof-of-principle, we applied the newly established method and found that a previously uncharacterized Arabidopsis thaliana tryptophan AT-related protein 1 is a highly promiscuous enzyme that can utilize 13 amino acid donors and three keto acid acceptors. These results demonstrate that this oxime-mass spectrometry imaging AT assay enables high-throughput discovery and comprehensive characterization of AT enzymes, leading to an accurate understanding of the nitrogen metabolic network.

Multiplex Lysosomal Enzyme Activity Assay on Dried Blood Spots Using Tandem Mass Spectrometry.

Deficiencies of the enzymes in lysosomes result in the accumulation of undegraded materials and subsequently cellular dysfunction. Early identification of deficiencies can lead to better clinical outcomes before irreversible organ and tissue damages occur. In this chapter, lysosomal enzymes are extracted from dried blood spots and incubated with the commercialized and multiplexed enzyme cocktail containing corresponding substrates and internal standards. After incubation, the enzymatic reactions are quenched, and the mixtures of the reaction products are prepared using liquid/liquid extractions. Multiple enzymes are quantified simultaneously using selected ion monitoring on liquid chromatography-mass spectrometry (LC-MS/MS) system.

Enzyme-based digital bioassay technology - key strategies and future perspectives.

Digital bioassays based on single-molecule enzyme reactions represent a new class of bioanalytical methods that enable the highly sensitive detection of biomolecules in a quantitative manner. Since the first reports of these methods in the 2000s, there has been significant growth in this new bioanalytical strategy. The principal strategy of this method is to compartmentalize target molecules in micron-sized reactors at the single-molecule level and count the number of microreactors showing positive signals originating from the target molecule. A representative application of digital bioassay is the digital enzyme-linked immunosorbent assay (ELISA). Owing to their versatility, various types of digital ELISAs have been actively developed. In addition, some disease markers and viruses possess catalytic activity, and digital bioassays for such enzymes and viruses have, thus, been developed. Currently, with the emergence of new microreactor technologies, the targets of this methodology are expanding from simple enzymes to more complex systems, such as membrane transporters and cell-free gene expression. In addition, multiplex or multiparametric digital bioassays have been developed to assess precisely the heterogeneities in sample molecules/systems that are obscured by ensemble measurements. In this review, we first introduce the basic concepts of digital bioassays and introduce a range of digital bioassays. Finally, we discuss the perspectives of new classes of digital bioassays and emerging fields based on digital bioassay technology.

Metal-Organic Frameworks-Based Immobilized Enzyme Microreactors Integrated with Capillary Electrochromatography for High-Efficiency Enzyme Assay.

Enzyme assays are important for studying enzyme-mediated biochemical reactions and for clinical diagnosis and drug development. The technique of an immobilized enzyme microreactor (IMER) integrated with capillary electrophoresis (CE) has been frequently utilized in online enzyme assays. However, the traditional approaches for IMER-CE enzyme analysis have some defects such as low loading capacity and poor stability. Herein, metal-organic frameworks (MOFs), which have enormous potential in the fields of enzyme immobilization and capillary electrochromatographic (CEC) separation, were first explored as novel support materials with good enzyme immobilization performance and stationary phases with excellent separation abilities to construct an integrated MOFs-IMER-CEC microanalysis system for a high-efficiency online enzyme assay. As a proof-of-concept demonstration, acetylcholinesterase (AChE) was immobilized on a densely packed UiO-66-NH2 nanocrystal coating on a capillary inner surface with abundant intercrystalline mesoporosity and was employed as a highly effective and robust IMER for CEC-integrated online enzyme analysis. The excellent separation performance of the UiO-66-NH2-modified capillary was verified by high-efficiency separation of three types of neutral, acidic, and basic compounds. The Michaelis-Menten constant and enzyme inhibition kinetics of UiO-66-NH2-IMER were systematically assessed, exhibiting distinct advantages such as remarkably increased enzyme loadability, superior affinity for substrates, and greatly improved stability and repeatability compared to CE-integrated IMERs prepared by the traditional covalent bonding method. Furthermore, the developed method was successfully utilized for detecting organophosphorus pesticides in leguminous vegetable samples, demonstrating its strong practicality. The study not only proposed a novel support material and construction strategy for a high-performance microchannel-based IMER but also can be widely used in bioanalysis and biosensing research.

Improved SARS-CoV-2 main protease high-throughput screening assay using a 5-carboxyfluorescein substrate.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a global threat to human health has highlighted the need for the development of novel therapies targeting current and emerging coronaviruses with pandemic potential. The coronavirus main protease (Mpro, also called 3CLpro) is a validated drug target against coronaviruses and has been heavily studied since the emergence of SARS-CoV-2 in late 2019. Here, we report the biophysical and enzymatic characterization of native Mpro, then characterize the steady-state kinetics of several commonly used FRET substrates, fluorogenic substrates, and six of the 11 reported SARS-CoV-2 polyprotein cleavage sequences. We then assessed the suitability of these substrates for high-throughput screening. Guided by our assessment of these substrates, we developed an improved 5-carboxyfluorescein-based FRET substrate, which is better suited for high-throughput screening and is less susceptible to interference and false positives than existing substrates. This study provides a useful framework for the design of coronavirus Mpro enzyme assays to facilitate the discovery and development of therapies targeting Mpro.

Metal-Nanoparticle-Supported Nanozyme-Based Colorimetric Sensor Array for Precise Identification of Proteins and Oral Bacteria.

Convenient, precise, and high-throughput discrimination of multiple bioanalytes is of great significance for an early diagnosis of diseases. Array-based pattern recognition has proven to be a powerful tool to detect diverse analytes, but developing sensing elements featuring favorable surface diversity still remains a challenge. In this work, we presented a simple and facile method to prepare programmable metal-nanoparticle (NP)-supported nanozymes (MNNs) as artificial receptors for the accurate identification of multiple proteins and oral bacteria. The in situ reduction of metal NPs on hierarchical MoS2 on polypyrrole (PPy), which generated differential nonspecific interactions with bioanalytes, was envisaged as the encoder to break through the limited supply of the receptor's quantity. As a proof of concept, three metal NPs, i.e., Au, Ag, and Pd NPs, were taken as examples to deposit on PPy@MoS2 as colorimetric probes to construct a cross-reactive sensor array. Based on the principal component analysis (PCA), the proposed MNN sensor array could well discriminate 11 proteins with unique fingerprint-like patterns at a concentration of 250 nM and was sufficiently sensitive to determine individual proteins with a detection limit down to the nanomolar level. Remarkably, two highly similar hemoglobins from different species (hemoglobin and bovine hemoglobin) have been precisely identified. Additionally, five oral bacteria were also well separated from each other without cross-classification at the level of 107 CFU mL-1. Furthermore, the sensor array allowed effective discrimination of complex protein mixtures either at different molar ratios or with minor varying components. Most importantly, the blind samples, proteins in human serums, proteins in simulated body fluid environment, the heat-denatured proteins, and even clinical cancer samples all could be well distinguished by the sensor array, demonstrating the real-world applications in clinical diagnosis.

The challenge of determining lysyl oxidase activity: Old methods and novel approaches.

The lysyl oxidase (LOX) family of enzymes catalyze the oxidative deamination of lysine and hydroxylysine residues in collagen and elastin in the initiation step of the formation of covalent cross-linkages, an essential process for extracellular matrix (ECM) maturation. Elevated LOX expression levels leading to increased LOX activity is associated with diverse pathologies including fibrosis, cancer, and cardiovascular diseases. Different protocols have been so far established to detect and quantify LOX activity from tissue samples and cultured cells, all of them showing advantages and drawbacks. This review article presents a critical overview of the main features of currently available methods as well as introduces some recent technologies called to revolutionize our approach to LOX catalysis.