Naphthalene Diimide and Bis-heteroleptic Ru(II) Complex-Based Hybrid Molecule with 3-in-1 Functionalities.

Multipurpose applications of a newly developed homobimetallic Ru(II) complex, Ru-NDI[PF6]4, which incorporates 1,10-phenanthroline and triazole-pyridine ligands and linked via a (-CH2-)3 spacer to the reputed anion-π interacting NDI system, are described. Solution-state studies of the bimetallic complex, including EPR, PL, UV-vis, and NMR experiments, reveal two sequential one-electron transfers to the NDI unit, generating NDI·- and NDI2- in the presence of F- selectively. This process inhibits the primary electron transfer from Ru(II) to the NDI unit, thereby allowing the 3MLCT-based emission of the complex to be recovered, resulting in a corresponding ten-fold increase in luminescence intensity. DFT and TD-DFT computational studies further elucidate the experimentally observed absorption spectra of the complex. Secondly, CT-DNA binding studies with the complex are performed using various spectroscopic analyses such as UV-vis, PL, and CD. Comparative DNA binding studies employing EB and molecular docking reveal that the binding with CT-DNA occurs through both intercalative and groove binding modalities. Thirdly, the photocatalytic activities of the complex towards C-C, C-N, and C-O bond formation in organic cross-coupling reactions, including the amidation of α-keto acids to amines and the oxidation of alcohol to aldehydes, are also demonstrated.

Small molecule interactions with biomacromolecules: selective sensing of human serum albumin by a hexanuclear manganese complex - photophysical and biological studies.

A covalently bonded hexanuclear neutral complex, [Mn6(µ3-O)2(3-MeO-salox)6(OAc)2(H2O)4] (1), has been synthesized and characterized by single crystal X-ray diffraction analysis along with IR and HRMS studies. Complex 1 has been found to selectively interact with human serum albumin (HSA), a model transport protein. The interaction of 1 with HSA was investigated by monitoring the change in the absorbance value of HSA at λ = 280 nm with increasing concentration of 1. Likewise, fluorescence titrations were carried out under two conditions: (i) titration of a 5 µM solution of complex 1 with the gradual addition of HSA, showing a ∼9-fold fluorescence intensity enhancement at 424 nm, upon excitation at 300 nm; and (ii) upon excitation at 295 nm, titration of 5 µM HSA solution with the incremental addition of complex 1, showing a quenching of fluorescence intensity at 334 nm, with simultaneous development of a new emission band at 424 nm. A linear form of the Stern-Volmer equation gives KSV = 9.77 × 104 M-1 and the Benesi-Hildebrand plot yields the binding constant as KBH = 1.98 × 105 M-1 at 298 K. The thermodynamic parameters, ΔS°, ΔH°, and ΔG°, were estimated by using the van't Hoff relationship which infer the major contribution of hydrophobic interactions between HSA and 1. It was observed that quenching of HSA emission arises mainly through a dynamic quenching mechanism as indicated by the dependence of average lifetime 〈τ〉 on the concentration of 1. The changes in the CD (circular dichroism) spectral pattern of HSA in the presence of 1 clearly establish the variation of HSA secondary structure on interaction with 1. The most probable interaction region in HSA for 1 was determined from molecular docking studies which establish the preferential trapping of 1 in the subdomain IIA of site I in HSA and substantiated by the results of site-specific marker studies. Complex 1 was further evaluated for its antiproliferative effects in lung cancer A549 cells, which strictly inhibits the growth of the cells in both 2D and 3D mammospheres, indicating its potential application as an anticancer drug.

A microenvironment-sensitive red emissive probe with a large Stokes shift for specific recognition and quantification of serum albumin in complex biofluids and live cells.

Human serum albumin (HSA) is regarded as a useful biomarker for rapid medical diagnosis of various disorders mainly related to the kidneys and liver. Hence, it is crucial to identify and monitor the HSA level in complex biofluids (urine and blood samples) using a simple approach. Herein, we have designed and synthesized an intramolecular charge transfer (ICT) based environment-sensitive fluorescent molecular probe, (E)-2-(3-(2-(5-methoxy-1H-indol-3-yl)vinyl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile (DCI-MIN), that can selectively interact with HSA in PBS buffer solution and exhibit a ∼78-fold enhancement in fluorescence intensity with a significant Stokes shift (∼126 nm), which is important to avoid interference from the excitation light. The significant red fluorescence response can be attributed to the suppression of free intramolecular rotation of the DCI-MIN probe inside the hydrophobic binding cavity of HSA and the low polar microenvironment present within HSA. According to the 3σ/slope method, the detection limit was found to be 1.01 nM (0.0671 mg L-1) in aqueous solutions, which is significantly lower than the normal level of HSA in healthy urine and blood serum, indicating its high sensitivity. DCI-MIN has the ability to exhibit useful applications, including the detection and quantification of HSA concentration in complex biofluids (human urine and blood samples) as well as the imaging of serum albumin in living cells.

Small Molecule Interactions with Biomacromolecules: DNA Binding Interactions of a Manganese(III) Schiff Base Complex with Potential Anticancer Activities.

A manganese(III) complex, [MnIII(L)(SCN)(enH)](NO3)·H2O (1•H2O) (H2L = 2-((E)-(2-((E)-2-hydroxy-3-methoxybenzylidene-amino)-ethyl-imino)methyl)-6-methoxyphenol), has been synthesized and characterized by single-crystal X-ray diffraction analysis. The interaction of 1•H2O with DNA was studied by monitoring the decrease in absorbance of the complex at λ = 324 nm with the increase in DNA concentration, providing an opportunity to determine the binding constant of the 1•H2O-ct-DNA complex as 5.63 × 103 M-1. Similarly, fluorescence titration was carried out by adding ct-DNA gradually and monitoring the increase in emission intensity at 453 nm on excitation at λex = 324 nm. A linear form of the Benesi-Hildebrand equation yields a binding constant of 4.40 × 103 M-1 at 25 °C, establishing the self-consistency of our results obtained from absorption and fluorescence titrations. The competitive displacement reactions of dyes like ethidium bromide, Hoechst, and DAPI (4',6-diamidine-2'-phenylindole dihydrochloride) from dye-ct-DNA conjugates by 1•H2O were analyzed, and the corresponding KSV values are 1.05 × 104, 1.25 × 104, and 1.35 × 104 M-1 and the Kapp values are 2.16 × 103, 8.34 × 103, and 9.0 × 103 M-1, from which it is difficult to infer the preference of groove binding over intercalation by these DNA trackers. However, the molecular docking experiments and viscosity measurement clearly indicate the preference for minor groove binding over intercalation, involving a change in Gibbs free energy of -8.56 kcal/mol. The 1•H2O complex was then evaluated for its anticancer potential in breast cancer MCF-7 cells, which severely abrogates the growth of the cells in both 2D and 3D mammospheres, indicating its promising application as an anticancer drug through a minor groove binding interaction with ct-DNA.

A coumarin embedded highly sensitive nitric oxide fluorescent sensor: kinetic assay and bio-imaging applications.

Fluorescence spectroscopy is a significant bio-analytical technique for specific detection of nitric oxide (NO) and for broadcasting the in vitro and in vivo biological activities of this gasotransmitter. Herein, a benzo-coumarin embedded smart molecular probe (BCM) is employed for NO sensing through detailed fluorescence studies in purely aqueous medium. All the spectroscopic analysis and literature reports clearly validate the mechanistic insight of this sensing strategy i.e., the initial formation of 1,2,3,4-oxatriazole on treatment of the probe with NO which finally converted to its carboxylic acid derivative. This oxatriazole formation results in a drastic enhancement in fluoroscence intensity due to the photoinduced electron transfer (PET) effect. The kinetic investigation unveils the second and first-order dependency on [NO] and [BCM] respectively. The very low detection limit (16 nM), high fluorescence enhancement (123 fold) in aqueous medium and good formation constant (Kf = (4.33 ± 0.48) × 104 M-1) along with pH invariability, non-cytotoxicity, biocompatibility and cell permeability make this probe a very effective one for tracking NO intracellularly.

Correction: A smart molecular probe for selective recognition of nitric oxide in 100% aqueous solution with cell imaging application and DFT studies.

Correction for 'A smart molecular probe for selective recognition of nitric oxide in 100% aqueous solution with cell imaging application and DFT studies' by Ananya Dutta et al., Org. Biomol. Chem., 2019, DOI: 10.1039/c9ob00177h.

Dansyl-appended CuII-complex-based nitroxyl (HNO) sensing with living cell imaging application and DFT studies.

We introduce herein, a novel copper complex-based fluorescent probe [CuII(DQ468)Cl]+ that exhibits a significant fluorescence turn-on response towards nitroxyl (HNO) with high selectivity over other biological reactive oxygen, nitrogen and sulfur species, including nitric oxide (NO). A smart strategy, involving HNO-induced reduction of paramagnetic [CuII(DQ468)Cl]+ to diamagnetic [CuI(DQ468)]+ with concomitant fluorescence enhancement via a PET mechanism is focused here. This reduction-based strategy was also supported by X-band EPR response and mass spectroscopy. The metal free probe (DQ468) showed high affinity towards Cu2+ to form [CuII(DQ468)Cl]+ with a 0.091 µM detection limit, which subsequently enabled the detection of HNO in an organo-aqueous medium at biological pH (7.4) in the green wavelength region (λem = 543 nm) with a LOD of 0.41 µM. The ground-state geometries of DQ468, [CuII(DQ468)Cl]+ and [CuI(DQ468)]+ were optimized by DFT calculations, which revealed that the central metal ion in [CuII(DQ468)Cl]+ is in a distorted tetrahedral geometry with the C1 point group. Additionally, the negligible cytotoxicity and good biocompatibility make the developed probe useful for the in vitro detection of HNO.

A smart molecular probe for selective recognition of nitric oxide in 100% aqueous solution with cell imaging application and DFT studies.

Herein, a simple, least-cytotoxic as well as an efficient fluorescent sensor HqEN480 was prepared from (quinolin-8-yloxy)-acetic acid ethyl ester (L1) and N,N-dimethylethylene diamine to recognize NO in 100% aqueous solution. Its marked selectivity and sensitivity towards NO, makes it a highly suitable probe for nitric oxide under in vitro conditions with the possibility of in vivo monitoring of NO. Upon addition of 3.5 equivalents of NO, there is an approximately 7 fold enhancement in fluorescence intensity in aqueous solution with a corresponding Kf value of (1.75 ± 0.07) × 104 M-1. Quantum yields of HqEN480 and [HqEN480-NO] compounds are determined to be 0.04 and 0.22, respectively, using acidic quinine sulphate as a standard. In terms of the 3σ method, the LOD for nitric oxide was found to be 53 nM thus, making the probe suitable for tracking NO in biological systems.

Phenazine-Embedded Copper(II) Complex as a Fluorescent Probe for the Detection of NO and HNO with a Bioimaging Application.

We report a novel phenazine-embedded fluorescent probe (2-[2-(pyridin-2-ylmethoxy)-phenyl]-1H-imidazo[4,5-b]phenazine, PIP), which upon complexation with Cu(II)-ion-forming [(PIP)CuII(Cl)] becomes nonfluorescent but regenerates fluorescence in a selective reaction with NO and HNO over different biologically reactive oxygen and nitrogen (ROS/RNS) species under physiological conditions. The fluorescence intensity of PIP gets quenched due to the formation of the [(PIP)CuII(Cl)] complex, which regenerates the fluorescence by 67 and 84% upon reaction either with NO or HNO, respectively, in the presence of other biological reducing species. Details of photophysical properties of PIP, [(PIP)CuII(Cl)], and [(PIP)CuI] have been studied by density functional theory (DFT) calculations. The recognition efficacy of [(PIP)CuII(Cl)] for exogenous and endogenous NO and HNO in A549 and RAW 264.7 cells with the flow cytometry application has also been demonstrated successfully.

Site-Selective Interaction of Human Serum Albumin with 4-Chloro-7-nitro-1,2,3-benzoxadiazole Modified Olanzapine Derivative and Effect of ß-Cyclodextrin on Binding: In the Light of Spectroscopy and Molecular Docking.

Here, we present a detailed investigation on the interaction of 4-chloro-7-nitro-1,2,3-benzoxadiazole (NBD) embedded olanzapine derivative (OLA-NBD) with a model transport protein, human serum albumin (HSA). The thermodynamic parameters, ΔHo, ΔSo, and ΔGo, as evaluated by considering the van't Hoff relationship imply the major contribution of electrostatic/ionic interactions for the HSA-OLA-NBD association. The OLA-NBD induced quenching of HSA emission occurs through static quenching mechanism, indicating a 1:1 association, as portrayed from Benesi-Hildebrand plot, with ∼104 M-1 association constant value, and it is in good harmony with the value estimated from anisotropy experiment. The invariance of the time-resolved decay behavior of HSA with added OLA-NBD concentration, along with matching dependency of the binding constant (Kb) value on temperature, also supports the occurrence of static quenching. The effect of ß-cyclodextrin on HSA-OLA-NBD binding is characterized by a smaller Kb value revealing that the OLA-NBD molecules are gradually removed from ß-CD by HSA to achieve its medicinal outcome of drug delivery. The outcome from circular dichroism (CD) illustrates the variation of HSA secondary structure upon interaction with OLA-NBD. Concurrently, HSA-OLA-NBD association kinetics is also explored by applying the fluorescence technique. The possible interaction zone of OLA-NBD in HSA is investigated from AutoDock-based docking simulation study.

Nitric Oxide Sensing through 1,2,3,4-Oxatriazole Formation from Acylhydrazide: A Kinetic Study.

A simple molecular probe displays highly selective turn-on response toward NO by the unprecedented NO-induced formation of a 1,2,3,4-oxatriazole ring exhibiting no interference from various endogenous biomolecules including DHA, AA, etc. Kinetics of the reactions between NO and the probe provide a mechanistic insight into the formation of 1,2,3,4-oxatriazole which showed that, though initially 1,2,3,4-oxatriazole is formed and extractable in solid form, it exists in equilibrium with the ring opened azide form which ultimately hydrolyzed and converted to carboxylic acid and nitrate. The reaction displays second-order dependence on [NO] and first-order on [Probe]. The probe is water-soluble, cell permeable, and noncytotoxic and appropriates for live cell imaging. This constitutes the first report where there is a direct evidence of NO-induced ring closing reaction of an acyl hydrazide moiety leading to the formation of 1,2,3,4-oxatriazole.

A dual response fluorescent sensor for HNO and S2- ions using a Cu(ii) complex based probe assisted by detailed DFT studies.

A Cu(ii) based sensor (1) prepared by the complexation between (quinolin-8-ylamino)-acetic acid hydrazide (L2) and Cu2+ ions has been developed for a highly sensitive and selective recognition of HNO and S2- over other biologically abundant anions with prominent enhancement in absorption and emission intensities. The sensor (1) shows weak fluorescence due to ET (electron transfer) but upon addition of HNO and S2- a large enhancement in the fluorescence intensity (F.I.) was observed over other possible competitive anions on the basis of reduction of Cu(ii) to Cu(i) and formation of CuS, respectively. The 1 : 1 complexation was characterized by mass spectrometry (MS), elemental analysis and Job's plot. The corresponding Kf value was evaluated to be (4.934 ± 0.05) × 104 M-1 for Cu2+ from UV-Vis absorption titration. Quantum yields of L2 and [Cu-L2 + S2-] and [Cu-L2 + HNO] complexes in acetonitrile (CH3CN) are found to be 0.107, 0.09 and 0.07, respectively, using quinine sulphate as the standard.

Selective sensing of nitric oxide by a 9,10-phenanthroquinone-pyridoxal based fluorophore.

In this article, we have designed and synthesized a new, convenient and efficient phenanthroquinone-pyridoxal based fluorogenic probe PQPY, highly suitable for the selective and sensitive detection of nitric oxide in an aerated aqueous (7 : 3/H2O : MeCN) medium at pH 7.0 (10 mM HEPES buffer). Upon addition of nitric oxide, this probe exhibits emission in the green region (λem = 505 nm) which is ascribed to ICT (intramolecular charge transfer) from the phenanthroquinone moiety to the imidazole -N-N[double bond, length as m-dash]O fragment. The apparent formation constant, Kf, of the NO product of the ligand is (1.00 ± 0.2) × 105 M-1 and the LOD is 78 nM. The substantial enhancement of the life-time of the ligand (τ0 = 2.68 ns) occurs due to binding with nitric oxide (τ0 = 3.96 ns). This probe is low cytotoxicity, cell permeable and suitable for living cell imaging application.

A rhodamine-based turn-on nitric oxide sensor in aqueous medium with endogenous cell imaging: an unusual formation of nitrosohydroxylamine.

A new sensor (L3) based on Rhodamine-B-en (2) and 2-(pyridin-2-ylmethoxy)benzaldehyde (1) has been developed for highly sensitive and selective recognition of NO in purely aqueous medium where the reaction of NO with the fluorophore leads to an unusual formation of nitrosohydroxylamine with the selective opening of the spirolactam ring over different cations, anions, amino-acids and other biological species with prominent enhancement in absorption and emission intensities. A large enhancement of fluorescence intensity for NO (11 fold) was observed upon addition of 3 equivalents of NO into the sensor in aqueous HEPES buffer (20 mM) at pH 7.20, µ = 0.05 M NaCl with naked eye detection. The corresponding Kf value was evaluated to be (7.55 ± 2.04) × 104 M-1 from the fluorescence titration plot. Quantum yields of L3 and the [L3 + NO] compound are found to be 0.07 and 0.77, respectively, using Rhodamine-6G as the standard. The LOD for NO was determined by the 3σ method and found to be 83.4 nM. The L3 sensor has low cytotoxicity, and is cell permeable and suitable for in vitro NO sensing. The in vivo compatibility of the sensor was also checked on zebrafish.

A turn-on fluorogenic chemosensor for Fe3+ and a Schottky barrier diode with frequency-switching device applications.

A novel highly sensitive and selective fluorescent chemosensor L has been synthesized and characterized by various physicochemical techniques. In 3 : 7 water : MeCN (v/v) at pH 7.2 (10 mM HEPES buffer, µ = 0.05 M LiCl), it selectively recognizes Fe3+ through 1 : 1 complexation resulting in a 106-fold fluorescence enhancement and a binding constant of 8.10 × 104 M-1. The otherwise non-fluorescent spirolactam form of the probe results a dual-channel (absorbance and fluorescence) recognition of Fe3+via CHEF (chelation enhanced fluorescence) through the opening of the spirolactam ring. We have also carried out fluorescence titration and anisotropy (r) studies in pure water in the presence of SDS (sodium dodecyl sulphate). Based on the dependence of FI (fluorescence intensity) and r on [SDS] it was proposed that the probe is trapped between two SDS monolayers which again interact among themselves by ππ stacking. As a result, there is an increase in FI up to [SDS] ∼ 7 mM - a phenomenon reminiscent of aggregation-induced enhancement of emission (AIEE). Beyond this concentration of SDS (7 mM), micelle formation takes place and the ππ stacked polymer now becomes a monomer and is trapped inside the micellar cavity. As a result, there is a decrease in FI at [SDS] > 7 mM. But for anisotropy, it increases with [SDS] beyond 7 mM. Ligand, metal, and SDS interactions are well established through different optical and morphological studies. [L-Fe(NO3)]2+ thin films on FTO (Fluorine-doped Tin Oxide) glass substrates have been designed with the help of the spin-coating deposition technique. The deposited film of thickness 1.6 × 10-5 cm is well characterized by optical band gap calculation with a direct band gap, εg ∼ 1.6 eV. FESEM was also performed for the [L-Fe(NO3)]2+/FTO film. The current-voltage characteristics were measured by the two-probe technique. Light-dependent exciton generation was carried out by taking the top and bottom contacts with graphite paste on FTO and on the [L-Fe(NO3)]2+ films for the measurement of switching behavior. The response ratio curve for the light-induced frequency-switching phenomena has been obtained. The frequency taped here is the oscillation frequency of the photo-generated electron and the hole in an exiton. Thus, the light-induced frequency-switching behavior and Schottky barrier diode characteristics of the material were established.

A hydrazone based probe for selective sensing of Al(iii) and Al(iii)-probe complex mediated secondary sensing of PPi: framing of molecular logic circuit and memory device and computational studies.

A hydrazone-based conjugate Nap-hyz-pyz (H3L3) with potential N2O2 donor atoms was found to act as a dual channel (colori- and fluori-metric) sensor towards Al3+ and PPi in H2O-MeOH (6 : 4, v/v) at pH 7.2 (40 mM HEPES buffer) at 25 °C. The formation constants, Kf = (3.49 ± 1.77) × 104 and (3.78 ± 0.1) × 104 M-1, of the sensor towards Al3+ were determined by absorption and fluorescence titrations, respectively. The 1 : 1 stoichiometry of the reaction was determined by Job's method and confirmed by ESI-MS+ (m/z) studies. The LOD for Al3+, as determined by the 3σ method, was found to be 114.54 nM. Most strikingly, the addition of ∼115 µM PPi to the Nap-hyz-pyz-Al3+ ensemble (20 µM ligand and 74 µM Al3+) leads to complete quenching of fluorescence. The fluorescence response of Nap-hyz-pyz towards Al3+ was not perturbed by the presence of 5 equivalents or more of other ions and inorganic anions. The structure of the [Al(L3)(H2O)] complex was delineated by DFT calculations. TD-DFT studies were performed to investigate various spectral transitions. Based on changes in the fluorescence intensities of Nap-hyz-pyz in the presence of Al3+ and PPi at 487 nm, INHIBIT and molecular logic gates were constructed and interpreted. The probe was found to be bio-compatible and cell permeable with no or negligible cytotoxicity; thus, it provides a good opportunity for in vitro cell imaging studies of these ions. The presence of ATP or Pi did not interfere with the fluorescent detection of PPi. Thus, these evident and excellent sensing capabilities of Nap-hyz-pyz towards Al3+ and PPi were further scrutinized in HepG2 cell lines.

Design of a Pyrene Scaffold Multifunctional Material: Real-Time Turn-On Chemosensor for Nitric Oxide, AIEE Behavior, and Detection of TNP Explosive.

A dual-emission pyrene-based new fluorescent probe (N-(4-nitro-phenyl)-N'-pyren-1-ylmethyl-ene-ethane-1,2-diamine (PyDA-NP)) displays green fluorescence for nitric oxide (NO) sensing, whereas it exhibits blue emission in the aggregated state. The mechanism of nitric oxide (NO/NO+) sensing is based on N-nitrosation of aromatic secondary amine, which was not interfered by reactive oxygen species and reactive nitrogen species. The aggregation-induced enhancement of emission (AIEE) behaviors of the PyDA-NP could be attributed to the restriction of intramolecular rotation and vibration, resulting in rigidity enhancement of the molecules. The AIEE behavior of the probe was well established from fluorescence, dynamic light scattering, scanning electron microscopy, transmission electron microscopy, X-ray diffraction, optical fluorescence microscopy, and time-resolved photoluminescence studies. In a H2O/CH3CN binary mixture (8:2 v/v), the probe showed maximum aggregation with extensive (833-fold) increases in fluorescence intensity and high quantum yield (0.79). The aggregated state of the probe was further applied for the detection of nitroexplosives. It displayed efficient sensing of 2,4,6-trinitrophenol (TNP), corroborating mainly the charge-transfer process from pyrene to a highly electron-deficient TNP moiety. Furthermore, for the on-site practical application of the proposed analytical system, a contact-mode analysis was performed.

A Smart Molecule for Selective Sensing of Nitric Oxide: Conversion of NO to HSNO; Relevance of Biological HSNO Formation.

A smart molecule, QT490, containing thiosemicarbazide moiety acts as a highly selective turn-on in vitro NO sensor through the unprecedented NO-induced transformation of thiosemicarbazide moiety to 1,3,4-oxadiazole heterocycle with the concomitant release of HSNO, thereby eliminating any interference from various endogenous biomolecules including dehydroascorbic acid, ascorbic acid, etc. The kinetic studies of the reactions between QT490 and NO provide a mechanistic insight into formation of HSNO/RSNO from the reaction between H2S/RSH and NO in the biological system. This novel probe is non-cytotoxic, cell permeable, water-soluble, and appropriate for intracellular cytoplasmic NO sensing with the possibilities of in vivo applications.

Di-oxime based selective fluorescent probe for arsenate and arsenite ions in a purely aqueous medium with living cell imaging applications and H-bonding induced microstructure formation.

A 2-hydroxy-5-methyl-benzene-1,3-dicarboxaldehyde di-oxime based turn-on blue emission fluorescent probe was found to recognize both AsO2(-) and H2AsO4(-) in a purely aqueous medium in intra and extra-cellular conditions. Self-organization of the ligand in the absence and presence of AsO2(-) and H2AsO4(-) was investigated by DLS, optical microscopy, optical fluorescence microscopy and FE-SEM methods.