Kinetics Of Complex Formation Research Articles

Kinetics and Mechanism of Formation and Stabilization of the RNA Polymerase-Promoter Open Compex

Specific binding of E. coli RNA polymerase holoenzyme (RNAP; α2ββ’σ70) to promoter DNA sets in motion a series of conformational changes that advance the initial closed complex (RPc), open 12-14 bp including the −10 region and the transcription start site, and at some promoters then stabilize the initial open complex dramatically. Our research focuses on the kinetics and mechanism of these conformational changes at different promoters, using Forster resonance energy transfer (FRET) and protein induced fluorescence enhancement (PIFE) together with fast footprinting and filter binding assays to determine the kinetics of open complex formation and dissociation, and to obtain structural and thermodynamic information about key intermediates. FRET and PIFE results to date demonstrate the importance of wrapping of upstream promoter DNA on RNAP for efficient bending of the downstream duplex into the active site cleft to form the advanced closed complex which is opened by RNAP in the rate limiting step. The initial open complex is found to be unstable, and is subsequently stabilized at some but not all promoters by conformational changes and interactions of in-cleft and downstream mobile elements of RNAP, directed by the discriminator sequence of the promoter. Dissociation kinetic studies with PIFE, FRET and filter binding assays are being used to probe the mechanism of these conformational changes in open complex stabilization. This project is funded by NIH support(GM103061).

Analysis of antimony species – lessons learnt from more than two decades of environmental research

Environmental contextThe environmental behaviour and toxicological effects of antimony depend strongly on the specific form of the element, and thus methods have been developed for measuring the various forms of antimony. These methods, applicable to quite clean samples, often fail when applied to more complex environmental samples. We discuss some of the pitfalls in determining environmental antimony forms and the resulting risk of getting the bigger picture wrong regarding antimony pollution. AbstractThe major findings of ~20 years of research on the analysis of antimony species in environmental samples are summarised in this paper. The complex chemistry of antimonite (SbIII) as well as of antimonate (SbV) plays a major role in chromatographic speciation of these species. For simple matrices, like surface or ground-water samples, antimony redox speciation has become a routine analysis and is robust and highly reproducible, if certain aspects are taken into consideration. These aspects are the formation of a stable complex of SbIII and complex formation kinetics. Then the antimony redox species can be separated on an anion-exchange column and detected with a suitable element detector (inductively coupled plasma–mass spectrometry (ICP-MS) or hydride generation–atomic fluorescence spectrometry (HG-AFS)) for trace analysis. The influence of complexing agents in the sample matrix, or in the eluent, on the formation of SbIII and SbV complexes and possible corruption of chromatography is discussed. This ability of antimony to form rather stable complexes also increases the risk of artefact formation during extraction of solid samples.

Phase separation and composition of coacervates of lactoferrin and caseins

The bovine milk casein proteins (αs-casein (ACN), β-casein (BCN) and κ-casein (KCN)) form complexes with lactoferrin (LF), an anionic protein also present in bovine milk that has an important bacteriostatic function especially in infant food. LF and each casein form complexes at near neutral pH, which is in between the respective pI's (≈8.3 and ≈ 4.6). The stoichiometry of the complexes is determined by the net charge of the proteins. Optimum complexation occurs at charge neutrality and is characterized by a maximum in turbidity. Mixing LF with each casein at a ratio where charge neutrality is obtained leads to a new complex coacervate phase. The kinetics of complex formation for LF/BCN and LF/KCN is rapid and appears to occur through a nucleation and coalescence process. However, the kinetics of complex formation between LF and ACN is much slower and therefore a nucleation and growth process is proposed, based on model calculations of the turbidity. The composition of the complexes is the same as the experimental mixing ratio if mixing ratio leads to a neutral complex. On standing and light centrifugation a complex-coacervate phase is formed, which is a viscous liquid.

Stopped Flow Kinetics of MnII Catalysed Periodate Oxidation of 2, 3- dimethylaniline - Evaluation of Stability Constant of the Ternary Intermediate Complex

The formation of ternary intermediate unstable complex during the oxidation of aromatic amines by periodate ion catalysed by MnII has been proposed in case of some anilines. This paper is the first report on stopped-flow kinetic study and evaluation of stability constant of ternary complex forming in the MnII - catalysed periodate oxidation of 2, 3-dimethylaniline (D) in acetone-water medium. Stop-flow spectrophotometric method was used to study the ternary complex formation and to determine its stability constant. The stop-flow trace shows the reaction to occur in two steps. The first step, which is presumably the formation of ternary complex, is relatively fast while the second stage is relatively quite slow. The stability constant evaluated for D - MnII - IO4- ternary complex by determining equilibrium absorbance is (2.2 ± 1.0) × 105. Kinetics of ternary complex formation was defined by the rate law(A) under pseudo first order conditions.ln{[C2]eq / ( [C2]eq -[C2])} = kobs . t (A)where, kobs is the pseudo first order rate constant, [C2] is concentration of ternary complex at given time t, and [C2]eq is the equilibrium concentration of ternary complex.

Protein-Based Microencapsulation with Freeze Pretreatment: Spray-Dried Oil in Water Emulsion Stabilized by the Soy Protein Isolate–Gum Acacia Complex

Spray-dried microparticles with lipid cores that dissolve β-carotene were prepared using a technique based on the complexation of soy protein and gum acacia. A freeze-pretreatment was applied to modify the resultant properties of the dried particles. An oil in water emulsion stabilized by soy protein isolate was mixed with gum acacia. The pH of the solution was adjusted to a selected value; this solution was frozen and thawed under controlled thermal protocols, then spray-dried to obtain the final products. Complexation was induced on the surface of the oil droplets owing to electrostatic interactions that occur below the isoelectric point (ca. pH 4.0) of the employed soy protein isolate. The freeze-pretreatment prior to spray-drying was expected to induce this interaction by freeze concentration and to control the kinetics of complex formation. When solutions with a pH of 4.0 and 3.0 were spray-dried, the encapsulation of β-carotene had similar efficiencies. The freeze-pretreatment was observed to have a significant effect on the specimens prepared from the solution with a pH of 4.0. Slow freezing conditions successfully improved the encapsulation efficiency and simultaneously reduced the amount of surface oil. Moreover, this pretreatment augmented the mass transfer resistance of the shell matrix, so that the release rate of dissolved β-carotene in the lipid core could be retarded.

Visualizing the molecular recognition trajectory of an intrinsically disordered protein using multinuclear relaxation dispersion NMR.

Despite playing important roles throughout biology, molecular recognition mechanisms in intrinsically disordered proteins remain poorly understood. We present a combination of (1)H(N), (13)C', and (15)N relaxation dispersion NMR, measured at multiple titration points, to map the interaction between the disordered domain of Sendai virus nucleoprotein (NT) and the C-terminal domain of the phosphoprotein (PX). Interaction with PX funnels the free-state equilibrium of NT by stabilizing one of the previously identified helical substates present in the prerecognition ensemble in a nonspecific and dynamic encounter complex on the surface of PX. This helix then locates into the binding site at a rate coincident with intrinsic breathing motions of the helical groove on the surface of PX. The binding kinetics of complex formation are thus regulated by the intrinsic free-state conformational dynamics of both proteins. This approach, providing high-resolution structural and kinetic information about a complex folding and binding interaction trajectory, can be applied to a number of experimental systems to provide a general framework for understanding conformational disorder in biomolecular function.

"Turn-on" protein fluorescence: in situ formation of cyanine dyes.

Protein reengineering of cellular retinoic acid binding protein II (CRABPII) has yielded a genetically addressable system, capable of binding a profluorophoric chromophore that results in fluorescent protein/chromophore complexes. These complexes exhibit far-red emission, with high quantum efficiencies and brightness and also exhibit excellent pH stability spanning the range of 2–11. In the course of this study, it became evident that single mutations of L121E and R59W were most effective in improving the fluorescent characteristics of CRABPII mutants as well as the kinetics of complex formation. The readily crystallizable nature of these proteins was invaluable to provide clues for the observed spectroscopic behavior that results from single mutation of key residues.

Title Monitoring Protamine‐Heparin Interactions Using Microcapillary Impedimetric Sensor

AbstractWe report an impedimetric sensor based on an interdigitated electrode array with electrode digits located at the bottom of microcapillaries formed in silicon dioxide which is used to monitor protamine‐heparin interactions. Modification of the active sensor surface with protamine, a cationic protein used as a high affinity heparin antagonist, permits to register protamine‐heparin complex formation as changes in the surface conductivity determined from the sensor impedance measured in an electrolyte solution. The adsorption curve shows that the detection limit is close to 0.01 U/mL (50 ng/mL) of heparin. The kinetics of the protamine‐heparin complex formation is rather slow and requires at least 15 min to be accomplished. Heparin reaction with protamine is irreversible, however, it is possible to renew the sensor surface putting it in contact with protamine solution and thus use the sensor several times. The future prospects for the proposed sensing strategy are quite wide and can include the grafting of the sensor surface with modified nanoparticles, magnetic particles or self‐assembled structures based on small synthetically‐accessible molecular‐scale building blocks which can act as protamine mimetics.

First stage of CoSi2 formation during a solid-state reaction

The kinetics of CoSi2 formation via a solid-state reaction between CoSi and single crystal Si has been the object of many studies in the past. Because of the importance of nucleation, complex kinetics has been reported. In this work, we investigate CoSi2 formation kinetics with in-situ diffraction during isothermal annealing of CoSi films on Si (100). In-situ measurements allow capturing the initial stage of CoSi2 formation. An initial t3/2 time-dependent evolution is observed and attributed to 3D growth of individual nuclei. This first regime is followed after the coalescence of the nuclei by a classical parabolic t1/2 one-dimensional film growth. We evidence a marked influence of the initial Co thickness (50 nm vs 10 nm) on the growth kinetics. A significant slowdown of the CoSi2 formation kinetics is observed for the thinnest film, whereas the activation energy remains the same. These results shine a new light on the complex formation kinetics of CoSi2 during solid-state reaction between CoSi and single crystal silicon and bring new knowledge about what occurs in the ultra-thin film regime, which is important for nanotechnologies.

Rotaxane Formation by Cucurbit[7]uril in Water and DMSO Solutions

The cucurbit[7]uril (CB7) host forms rotaxane-type complexes with dumbbell-shaped, cationic guests bis(3,5-dimethoxybenzyl)-4,4'-bipyridinium (1(2+)) and bis(3,5-diethoxybenzyl)-4,4'-bipyridinium (2(2+)). The kinetics of complex formation is slower with the latter guest because of its bulkier end groups. Rotaxane formation was found to be thermodynamically more favorable and kinetically faster in D2O than in DMSO-d6 solution, which highlights the importance of hydrophobic interactions in the assembly of cucurbituril complexes.

Abstract 1718: Schlafen 11 (SLFN11) is a critical determinant of cellular sensitivity to PARP inhibitors

Abstract Poly(ADP-ribose)polymerases (PARP) are DNA damage sensors and major repair factors for DNA single-strand breaks. Since the discovery of the synthetic lethality of PARP inhibitors in homologous recombination-deficient cells, the mechanism by which PARP inhibitors exert their cytotoxicity has been dominantly interpreted as an accumulation of unrepaired single-strand breaks resulting from catalytic PARP inhibition. We recently reported that several PARP inhibitors have an additional cytotoxic mechanism by trapping PARP-DNA complexes (1). BMN 673 is the most potent PARP inhibitor to date in terms of cytotoxicity and PARP trapping potency (2). Nevertheless, the NCI60 data show that half of the 60 cell lines are tolerant to BMN 673. To elucidate the mechanisms of tolerance, we searched for genes with expression levels correlated to BMN 673 sensitivity, and found that Schlafen 11 (SLFN11) is one of the most positively correlated genes. Most cells without SLFN11 expression were tolerant to BMN 673. To examine the involvement of SLFN11 in BMN 673 sensitivity, we disrupted the SLFN11 gene in DU145 prostate cancer cells and EW8 Ewing's sarcoma cells where SLFN11 expression levels and BMN 673 sensitivities are high. Knocking out SLFN11 in DU145 and EW8 did not affect their cell growth or cell cycle progression under normal conditions, but diminished their hypersensitivity to BMN 673 and another PARP inhibitor, olaparib. Cell cycle analyses revealed that SLFN11-proficient cells arrested at S phase while SLFN11 deficient cells were able to progress to G2 phase in the presence of BMN 673. Interestingly, arresting points at S phase in SLFN11 proficient cells were earlier when more PARP-DNA complexes were induced, suggesting that SLFN11 inhibits DNA replication under DNA damage. Differential kinetics of DNA repair and PARP-DNA complex formation were observed between SLFN11 proficient versus deficient cells. Our data suggest that the expression of SLFN11 may be a novel genomic biomarker for PARP inhibitor sensitivity. 1. Murai J, Huang S-yN, Das BB, Renaud A, Zhang Y, Doroshow JH, et al. Trapping of PARP1 and PARP2 by Clinical PARP Inhibitors. Cancer Research. 2012;72:5588-99. 2. Murai J, Huang SN, Renaud A, Zhang Y, Ji J, Takeda S, et al. Stereospecific PARP trapping by BMN 673 and comparison with olaparib and rucaparib. Mol Cancer Ther. 2014; Submitted. Citation Format: Junko Murai, Rozenn Josse, James H. Doroshow, Yves Pommier. Schlafen 11 (SLFN11) is a critical determinant of cellular sensitivity to PARP inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1718. doi:10.1158/1538-7445.AM2014-1718

Altered spatio-temporal dynamics of RNase H2 complex assembly at replication and repair sites in Aicardi-Goutières syndrome.

Ribonuclease H2 plays an essential role for genome stability as it removes ribonucleotides misincorporated into genomic DNA by replicative polymerases and resolves RNA/DNA hybrids. Biallelic mutations in the genes encoding the three RNase H2 subunits cause Aicardi-Goutières syndrome (AGS), an early-onset inflammatory encephalopathy that phenotypically overlaps with the autoimmune disorder systemic lupus erythematosus. Here we studied the intracellular dynamics of RNase H2 in living cells during DNA replication and in response to DNA damage using confocal time-lapse imaging and fluorescence cross-correlation spectroscopy. We demonstrate that the RNase H2 complex is assembled in the cytosol and imported into the nucleus in an RNase H2B-dependent manner. RNase H2 is not only recruited to DNA replication foci, but also to sites of PCNA-dependent DNA repair. By fluorescence recovery after photobleaching, we demonstrate a high mobility and fast exchange of RNase H2 at sites of DNA repair and replication. We provide evidence that recruitment of RNase H2 is not only PCNA-dependent, mediated by an interaction of the B subunit with PCNA, but also PCNA-independent mediated via the catalytic domain of the A subunit. We found that AGS-associated mutations alter complex formation, recruitment efficiency and exchange kinetics at sites of DNA replication and repair suggesting that impaired ribonucleotide removal contributes to AGS pathogenesis.

A Comparative Study for Chelation of Iron(II) and Iron(III) with Active Component of Antiparkinsonian Drug Molecule

Normal 0 In the present study interaction of Fe(II) and Fe(III) with antiparkinsonian drug molecule, Levodopa (LD), is investigated using Potentiometry and Spectrophotometry. Fe(II) and Fe(III) both metals showed same spectra, which is an evidence of identical complex. Molar absorptivities of the complexes were found to be more than 100 M -1 cm -1 , showed charge transfer spectra. Addition of an antioxidant, decolorized the initial intense color of the complex, which is evident of high oxidation state of iron in the complex. Catecholic ligands are strong reductants, thus chelate a metal ion in high oxidation state and show LMCT bands. These points confirm that, in the complex, iron has high oxidation state, regardless of initial source of metal. Stoichiometry of the complex was explored by Mole Ratio, Slope Ratio and Job’s Plot methods showed consistency in results. Fe(II) and Fe(III) both form 1:3 metal to ligand ratio. Formation constant, found by different methods, are high and consistent. pK 1 = 6.42, pK 2 = 6.60 and pK 3 = 13.81 for Fe(III)-LD complex and pK 2 = 7.21 and pK 3 = 9.94, rather than pK 1 which is unidentified, for Fe(II)-LD complex, were evaluated by Potentiometric data. Kinetics of complex formation showed the direct relationship in case of Fe(III)-LD, while in Fe(II)-LD complex the saturation pathway was observed. t 1/2 for Fe(II) and Fe(III) complex with LD were calculated, indicative of very fast reaction in case of Fe(III)-LD having t 1/2 = 0.917 ms. Kinetics results helps to originates mechanism of the reaction.

Formation of cytotoxic α-lactalbumin / sodium oleate complexes: Concentration and temperature effects

Cytotoxic complexes may be formed by mixing whey proteins and oleic acid or sodium oleate. The present study investigated the kinetics of complex formation when the whey protein α-lactalbumin (α-LA) is mixed with sodium oleate. Intrinsic fluorescence showed that the complex formed at room temperature during the mixing of α-LA and sodium oleate. However, some of the structural changes brought about by the binding of the sodium oleate can be reversed by dialysis, when loosely bound oleate is removed. During dialysis, solutions with lower protein concentrations had reduced buffering capacity, and so the pH decreased more; this decrease in pH was correlated with a loss of oleate from the complex. The cytotoxicity of all the samples produced in this study was strongly correlated with the amount of oleate bound in the complex, confirming previous studies that suggested that oleic acid is the cytotoxic agent in the complex.

ApoE and Aβ in Alzheimer’s Disease: Accidental Encounters or Partners?

Among the three human apolipoprotein E (apoE) isoforms, apoE4 increases the risk of Alzheimer's disease (AD). While transporting cholesterol is a primary function, apoE also regulates amyloid-β (Aβ) metabolism, aggregation, and deposition. Although earlier work suggests that different affinities of apoE isoforms to Aβ might account for their effects on Aβ clearance, recent studies indicate that apoE also competes with Aβ for cellular uptake through apoE receptors. Thus, several factors probably determine the variable effects apoE has on Aβ. In this Review, we examine biochemical, structural, and functional studies and propose testable models that address the complex mechanisms underlying apoE-Aβ interaction and how apoE4 may increase AD risk and also serve as a target pathway for therapy.

Synthesis, Characterization and Cytotoxicity of (η6‐p‐cymene)ruthenium(II) Complexes of α‐Amino Acids

AbstractArene ruthenium complexes of α‐amino acids, obtained by mixing aqueous solutions of [(η6‐p‐cymene)RuCl2]2 in the presence of AgCF3SO3 with various amino acids, have been studied at 37 °C using NMR spectroscopy and electrospray ionization mass spectrometry (ESI‐MS). Small hydrophobic α‐amino acid anions were found to form bidentate N,O‐chelate complexes, whereas α‐amino acids with coordinating side chains (e.g. serine, aspartic acid, histidine) acted as tridentate ligands. For many amino acids, more than two chelate complexes were formed. Presumably, complexes with the general formula [(η6‐p‐cymene)Ru(AA)2]n+ and bridged complexes with the general formula [{(η6‐p‐cymene)Ru}2(μ‐AA)2(μ‐OH)]+ were observed together with the expected bi‐ and tridentate chelate complexes. All complexes, isolated as triflate salts, were evaluated for their in vitro anticancer activity against the human ovarian cancer cell line A2780 and its cisplatin‐resistant mutant A2780cisR. All complexes were found to be highly cytotoxic, with IC50 values ranging from 0.16–19.8 μM. Interestingly, all complexes were selectivity active against A2780 cells relative to the A2780cisR variants, indicating a distinct mechanism of action that differs from that of many previously reported cytotoxic ruthenium complexes. No direct correlation between the kinetics of complex formation and cytotoxicity was apparent, suggesting that other physicochemical parameters such as complex stability and ligand exchange kinetics may play important roles in the biological activity.

Speciation dynamics of metals in dispersion of nanoparticles with discrete distribution of charged binding sites

We report a comprehensive theory to evaluate the kinetics of complex formation between metal ions and charged spherical nanoparticles. The latter consist of an ion-impermeable core surrounded by a soft shell layer characterized by a discrete axisymmetric 2D distribution of charged sites that bind metal ions. The theory explicitly integrates the conductive diffusion of metal ions from bulk solution toward the respective locations of the reactive sites within the particle shell volume. The kinetic constant k for outer-sphere nanoparticle-metal association is obtained from the sum of the contributions stemming from all reactive sites, each evaluated from the corresponding incoming flux of metal ions derived from steady-state Poisson-Nernst-Planck equations. Illustrations are provided to capture the basic intertwined impacts of particle size, overall particle charge, spatial heterogeneity in site distribution, type of particle (hard, core-shell or porous) and concentration of the background electrolyte on k. As a limit, k converges with predictions from previously reported analytical expressions derived for porous particles with low and high charge density, cases that correspond to coulombic and mean-field (smeared-out) electrostatic treatments, respectively. The conditions underlying the applicability of these latter approaches are rigorously identified in terms of (i) the extent of overlap between electric double layers around charged neighbouring sites, and (ii) the magnitude of the intraparticulate metal concentration gradient. For the first time, the proposed theory integrates the differentiated impact of the local potential around the charged binding sites amidst the overall particle field, together with that of the so-far discarded intraparticulate flux of metal ions.

CYCLIC VOLTAMMETRY OF THE SECOND ORDER CE MECHANISM AT THE THIN MERCURY FILM COVERED STATIONARY PLANAR ELECTRODE

A theoretical model of electrode reaction influenced by the kinetics of complex formation and dissociation is developed for cyclic voltammetry. It is assumed that the electro-reductions of free metal ions and the complex are reversible and totally irreversible, respectively, and that the ligand is not present in great excess. The results show that the stripping peak current and potential depend on the relative bulk concentration of ligand, particularly within the range 1 < c*L,tot / c*M.tot < 3, and on the rate constant of complex formation.

Molecular Mechanisms in the Activation of Abscisic Acid Receptor PYR1

The pyrabactin resistance 1 (PYR1)/PYR1-like (PYL)/regulatory component of abscisic acid (ABA) response (RCAR) proteins comprise a well characterized family of ABA receptors. Recent investigations have revealed two subsets of these receptors that, in the absence of ABA, either form inactive homodimers (PYR1 and PYLs 1–3) or mediate basal inhibition of downstream target type 2C protein phosphatases (PP2Cs; PYLs 4–10) respectively in vitro. Addition of ABA has been shown to release the apo-homodimers yielding ABA-bound monomeric holo-receptors that can interact with PP2Cs; highlighting a competitive-interaction process. Interaction selectivity has been shown to be mediated by subtle structural variations of primary sequence and ligand binding effects. Now, the dynamical contributions of ligand binding on interaction selectivity are investigated through extensive molecular dynamics (MD) simulations of apo and holo-PYR1 in monomeric and dimeric form as well as in complex with a PP2C, homology to ABA insensitive 1 (HAB1). Robust comparative interpretations were enabled by a novel essential collective dynamics approach. In agreement with recent experimental findings, our analysis indicates that ABA-bound PYR1 should efficiently bind to HAB1. However, both ABA-bound and ABA-extracted PYR1-HAB1 constructs have demonstrated notable similarities in their dynamics, suggesting that apo-PYR1 should also be able to make a substantial interaction with PP2Cs, albeit likely with slower complex formation kinetics. Further analysis indicates that both ABA-bound and ABA-free PYR1 in complex with HAB1 exhibit a higher intra-molecular structural stability and stronger inter-molecular dynamic correlations, in comparison with either holo- or apo-PYR1 dimers, supporting a model that includes apo-PYR1 in complex with HAB1. This possibility of a conditional functional apo-PYR1-PP2C complex was validated in vitro. These findings are generally consistent with the competitive-interaction model for PYR1 but highlight dynamical contributions of the PYR1 structure in mediating interaction selectivity suggesting added degrees of complexity in the regulation of the competitive-inhibition.

Kinetic and Stoichiometric Characterisation of Streptavidin‐Binding Aptamers

Aptamers are oligonucleotide ligands that are selected for high-affinity binding to molecular targets. Only limited knowledge relating to relations between structural and kinetic properties that define aptamer-target interactions is available. To this end, streptavidin-binding aptamers were isolated and characterised by distinct analytical techniques. Binding kinetics of five broadly similar aptamers were determined by surface plasmon resonance (SPR); affinities ranged from 35-375 nM with large differences in association and dissociation rates. Native mass spectrometry showed that streptavidin can accommodate up to two aptamer units. In a 3D model of one aptamer, conserved regions are exposed, strongly suggesting that they directly interact with the biotin-binding pockets of streptavidin. Mutational studies confirmed both conserved regions to be crucial for binding. An important result is the observation that the most abundant aptamer in our selections is not the tightest binder, emphasising the importance of having insight into the kinetics of complex formation. To find the tightest binder it might be better to perform fewer selection rounds and to focus on post-selection characterisation, through the use of complementary approaches as described in this study.