Zn-dependent β-amyloid Aggregation and its Reversal by the Tetrapeptide HAEE

The pathogenesis of Alzheimer's disease (AD) is associated with the formation of cerebral amyloid plaques, the main components of which are the modified Aβ molecules as well as the metal ions. Aβ isomerized at Asp7 residue (isoD7-Aβ) is the most abundant isoform in amyloid plaques. We hypothesized that the pathogenic effect of isoD7-Aβ is due to the formation of zinc-dependent oligomers, and that this interaction can be disrupted by the rationally designed tetrapeptide (HAEE). Here, we utilized surface plasmon resonance, nuclear magnetic resonance, and molecular dynamics simulation to demonstrate Zn2+-dependent oligomerization of isoD7-Aβ and the formation of a stable isoD7-Aβ:Zn2+:HAEE complex incapable of forming oligomers. To demonstrate the physiological importance of zinc-dependent isoD7-Aβ oligomerization and the ability of HAEE to interfere with this process at the organismal level, we employed transgenic nematodes overexpressing human Aβ. We show that the presence of isoD7-Aβ in the medium triggers extensive amyloidosis that occurs in a Zn2+-dependent manner, enhances paralysis, and shortens the animals’ lifespan. Exogenous HAEE completely reverses these pathological effects of isoD7-Aβ. We conclude that the synergistic action of isoD7-Aβ and Zn2+ promotes Aβ aggregation and that the selected small molecules capable of interrupting this process, such as HAEE, can potentially serve as anti-amyloid therapeutics.

transgenic mice overexpressing human Aβ, potentially acting as an amyloid seeding. Moreover, synthetic isoD7-Aβ, rather than intact Aβ, causes a significantly higher level of tau phosphorylation in cell culture [7]. These findings suggest the role of isoD7-Aβ as a molecular trigger for the pathogenic cascade of AD and a potential drug target. Indeed, it has been recently shown that targeting isoD7-Aβ with an antibody attenuates AD-like pathology in transgenic mice [8]. The pathological properties of isoD7-Aβ may be a result of its enhanced ability to form oligomers in the presence of zinc ions [9,10]. Zn 2+ promotes the accumulation of Aβ oligomers in synapses in a neuronal activity-dependent manner, and of all the divalent cations, it plays the most significant role in the formation of senile plaques [11,12]. However, there is no experimental data implicating the interaction of Zn 2+ and isoD7-Aβ in AD pathology.
In this study, we utilized the C. elegans model of Aβ amyloidosis to study the effect of isoD7-Aβ and zinc ions on animal pathophysiology and aging. We show that the concurrent administration of Zn 2+ and isoD7-Aβ leads to a significant increase in amyloidosis accompanied by the shortening of animals' lifespan. We further demonstrate that the tetrapeptide, HAEE, previously designed to counter the receptor toxicity of Aβ in vitro [13] , can prevent zinc-induced oligomerization of isoD7-Aβ, negate the pro-amyloid effects of isoD7-Aβ:Zn 2+ in live animals, and restore their lifespan. Our surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), and molecular dynamics (MD) studies indicate that the molecular mechanism underlying the anti-amyloid effect of HAEE relies on its specific zinc-dependent and stable binding to Aβ and isoD7-Aβ. Together, these results elucidate the fundamental role of non-covalent complexes between the zinc ion and isoD7-Aβ in triggering the pathological aggregation of endogenous Aβ molecules and suggest that the compounds targeting such complexes have a therapeutic potential.

SPR analysis
All SPR measurements were carried out at 25°C using an optical biosensor Biacore 8K (GE Healthcare, USA) with CM4 optical chip. Covalent immobilization of the peptide ligands on the surface of the optical chip was performed using the sulfhydryl group of the C-terminal tetraglycylcysteine tag. Briefly, carboxyl groups on the chip surface were activated by serial injections of mixture of 0.4 M EDC/0.1 M NHS at a flow rate of 10 μL/min for 2 min and a solution of 80 mM PDEA in 100 mM borate buffer (pH 8.5) for 4 min. Solutions of 2 μM peptides in the immobilization buffer (10 mM acetate buffer, pH 4.5) were injected for 2 min at a flow rate of 10 μL/min (three repeats). Unreacted activated groups on the chip were blocked by further injection of a solution containing 50 mM cysteine, 1 M NaCl, 0.1 M sodium acetate (pH 4.3) for 4 min at a flow rate of 5 μL/min. Possible nonspecific analyte binding to the chip surface was evaluated using a free (control) channel of the biosensor exposed to the same treatments as the working channel except peptides. SPR signals were recorded in real time in resonance units (RU; 1 RU corresponds to 1 pg of analyte) and were presented in the form of sensorgrams showing timedependent signal changes. A series of sensorgrams representing the difference of SPR-signals from working and control channels were obtained by serial injections of analyte solutions (Fig. 1B) through the working and control channels at a flow rate of 30 μL/min for 3 min. All SPR measurements were repeated 3 times. After each measurement the optical chip surface was regenerated by injecting an HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20) buffer for 30 s. Analyte samples were prepared in the running buffer (10 mM HEPES, pH 6.8 in the absence of Zn 2+ and 10 mM HEPES, 100 µM ZnCl2, pH 6.8). The obtained sensorgrams were analyzed with BIAevaluation v.4.1 software.

C and
Aging and Disease • Volume 14, Number 2, April 2023 311 15 N) pulsed field z gradient probe. 1D NMR spectra were processed and analyzed using the Mnova software (Mestrelab Research, Spain). In order to monitor peptidepeptide interactions NMR titration experiments were carried out. Peptides at a concentration of 0.2-2.0 mM at pH 6.8 were titrated with a solution of HAEE and ZnCl2 in a buffer of identical composition at the same pH value. 1D spectra were recorded for each titration point.

Modeling and MD simulations of the isoD7-Aβ:HAEE interactions
The templates for amyloid beta-peptide isoD7-Aβ16 and HAEE structure modeling with zinc were taken from the PDB:2MGT solution NMR structure of zinc-induced dimer of the human amyloid beta-peptide metal binding domain 1-16 with Alzheimer's disease pathogenic English mutation H6R [9]. To obtain the isoD7-Aβ16 structure, in one of the H6R-Aβ1-16 molecules in the PDB:2MGT dimer, Asp7 was replaced with iso-asparagine and Arg6 was replaced with histidine, thus, Aβ16 reverted to the iso-Asp7 primary structure. In the initial HAEE structure, hydrogens, acetyl, and amino (CH3CO and NH2 respectively) end groups were added and the resulting structure was minimized in water and relaxed for 100 ns using the Gromacs package [14]. HAEE was superposed with the structure of the second H6R-Aβ16 molecule in the PDB:2MGT dimer, so that the positions of arginine and histidine atoms of Aβ16 and HAEE, which coordinate zinc, corresponded as much as possible. This initial structure, after isoD7-Aβ16 was replaced by isoD7-Aβ42, was also used to model the isoD7-Aβ42:HAEE interaction. The structure of isoD7-Aβ42 was modeled by us previously [15]. Expert modeling was used to align the position of His13 side chain in isoD7-Aβ42 with the corresponding side chain in the template zinc coordination interface. The resulting structures were solvated with TIP3P water and NaCl ions at a concentration of 115 mM. To model a zinc ion, two different special force fields based on quantum calculations were used, with practically no difference in the resulting Aβ:HAEE structures [16,17]. The parametrization of the Aβ, HAEE, Glu, and His residues in coordinating zinc have been changed in accordance with the above force field. After 200 ps equilibration in NVT and NPT ensembles respectively the systems were simulated for 100 ns of MD production run at 300 K. All simulations were carried out using the particle mesh Ewald technique with repeating boundary conditions and 1 nm cut-offs, using the LINCS constraint algorithm with a 2 fs time step. The final structure after completion of MD simulation was taken as the representative structure.
To analyze the stability of the Aβ final conformations, root mean square distance (RMSD) and root mean square fluctuation (RMSF) calculations of the MD trajectories were performed. The RMSD plot shows conformational stability of the structure during the MD simulation relevant to the reference structure.

Treatment with isoD7-Aβ42 peptide and HAEE
Nematodes were purified from unwanted microflora by the alkaline hypochlorite method, grown to the L4 stage at 15°C on NGM plates with bacterial lawn. Approximately 140 animals were then placed in a drop of 0.2 ml M9-buffer +/-ZnSO4 (20μM) +/-isoD7-Aβ42 (40μM) +/-HAEE (4mM) and cultured for 4 hours at 25 o C by stirring. Further, all contents were transferred to Petri plates with NGM or NGMZn with OP50 and placed in a temperature-controlled incubator at 25 o C. Next day, worms were transferred onto similar media with a bacterial substrate and the lifespan was determined at 25°C (Supplementary Tables 3, 4).

Imaging and quantitative analysis of amyloid deposits
Nematodes were grown on NGM or NGMZn medium with a E. coli OP50 strain at 25°C and treated with amyloid peptides and/or HAEE as described above. At the A1 stage, the animals were stained with 1 mM X-34 (Sigma-Aldrich, St. Louis, MO, USA) in 10 mM Tris pH 8.0 for two hours. For de-staining, they were afterwards cultured for 16 hours on NGM or NGMZn with a bacterial lawn at the same temperature. Stained individual worms were mounted on glass slides in agar and anesthetized by incubation in 0.1% Na3N. Amyloid aggregates were visualized with Leica TCS SP5 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany) using 405 nm laser for excitation and a 430-570 bandpass emission filter with 63x oil immersion objective. Worms were imaged as z-stacks of 10-15 images 4.1 µm thickness each. For imaging, standard Las X software (Leica Microsystems GmbH, Wetzlar, Germany) with a plugin for confocal imaging was used. Acquired z-stacks were processed with Las X software to obtain maximum projections. Background subtraction and thresholding were performed by WCIF ImageJ with identical parameters for all images. Total plaque area, mean fluorescence intensity and plaques count were quantified for each worm in an area spanning from anterior end to posterior bulb. Data for plaque area is presented on graphs as individual values normalized by the amyloid load in control with bars representing sample means. The comparison of data groups was performed with ordinary one-way ANOVA. Post-hoc analysis was performed with the Tukey test. The Shapiro-Wilk test was used to confirm the normality of the dataset. Statistical analysis was performed with GraphPad Prism 9.1 software (GraphPad Software Inc., CA, USA). The representative nematode images for Fig. 2A were selected to have the plaques area that is close to the mean of each sample.

Paralysis assay
Synchronized eggs of CL2120 were grown to the L4 stage at 15°C on NGM plates with bacterial lawn, were treated with ZnSO4, Aβ42 (40 μM

Lifespan analysis
Lifespans were monitored at 25°C as described previously [20,21]. All experiments were repeated at least three times and ≈130 worms were used for each experiment. Details regarding repeated experiments and amounts of animals used for experiments are summarized in Supplementary Tables 3, 4. In all cases, stage L4 worms were used at t = 0 for lifespan analyses and worms were transferred every other day to new agar plates. Worms were judged as dead when they ceased pharyngeal pumping and did not respond to prodding with a platinum wire. Escaped animals or animals with internal hatching were not included in lifespan calculation. Data was analyzed and Boltzmann sigmoid survival curves generated using the SciDAVis statistical analysis software package. Mean lifespans were compared in Microsoft Excel using the Student t test, applying one-tailed distribution and two-sample equal variance [22]. All lifespan plots represent the composites of all independent experiments tabulated in Supplementary Tables 3, 4.
Mean percentage change ±SD of lifespan after treatment, relative to untreated control are indicated in each graph in the same color as the curve.

Statistical analysis
The applied statistical tests and software for each type of data are specified in the respective Methods subsections. For all of the data, before applying parametric statistical tests, the D'Agostino & Pearson normality test in GraphPad Prism 9.1 software was used to demonstrate that all sample data is distributed normally.

HAEE stably interacts with Aβ and interferes with its Zn 2+ -dependent dimerization
In contrast to normal Aβ, both the full-length isomerized Aβ, isoD7-Aβ42, and its metal-binding fragment, isoD7-Aβ16, stimulate amyloidosis in transgenic mice [23,24]. This correlates with the increased capacity of isoD7-Aβ42 and isoD7-Aβ16 for zinc-dependent aggregation [9]. We hypothesized that the contribution of isoD7-Aβ to the development of AD is based on the formation of zincbound complexes that promote pathological aggregation of Aβ (Fig. 1A). To test this hypothesis, we utilized a tool compound -the tetrapeptide HAEE, which was rationally designed to interact with the zinc-binding site of Aβ ( 11 EVHH 14 ) [13,25]. We first used SPR analysis to study the interaction between the immobilized Aβ42 or isoD7-Aβ42 and the soluble HAEE tetrapeptide. In the absence of Zn 2+ , no interaction of HAEE with immobilized amyloid peptides was detected (Supplementary Fig. 1). In the presence of 100 µM ZnCl2, the sensorgrams of the HAEE interaction with both Aβ42 or isoD7-Aβ42 were obtained (Fig. 1B) and the interaction parameters calculated (Supplementary Table 1). The KD values for HAEE-Aβ42 and HAEE-isoD7-Aβ42 complexes were estimated to be 4.1±0.3 µM and 10.4±0.4 µM, respectively.

Figure 1. Interaction of HAEE with betaamyloid. (A)
A model for the contribution of isoD7-Aβ to AD pathology, and the possible ways of preventing its deleterious effects. Aβ spontaneously converts into isoD7-Aβ, which is accumulated over age, likely due to reduced Aβ clearance. Isomerized Aβ promotes AD pathology, potentially via formation of Zn-bound complexes serving as the seeds for a generation of neurotoxic oligomers. Treatment with anti-isoAβ antibodies is a promising therapeutic strategy already showing effectiveness in AD model mice 9 . HAEE was shown to hamper amyloid accumulation in animal models of AD 17 , and its effect may be due to the formation of harmless isoAβ:Zn 2+ :HAEE complexes, which are subject to degradation. Next, we used NMR spectroscopy to obtain structural information on the interaction of HAEE with Aβ and isoD7-Aβ. Full-size Aβ quickly aggregates in solution in the presence of Zn 2+ [26]. Therefore, we used a metalbinding fragment of Aβ (1-16 aa), which is an established model to study the role of Zn 2+ in Aβ interactions . In the presence of Zn 2+ the HAEE-induced chemical shift of Aβ16 (Supplementary Fig. 2) and isoD7-Aβ16 ( Supplementary Fig. 3) signal was clearly observed. The presence of the tetrapeptide eliminated the signal at 0.2 ppm, which is characteristic of the dimeric form of the Aβ metal binding domain [9]. Thus, we conclude that HAEE inhibits the process of zinc-dependent dimerization in both Aβ16 and isoD7-Aβ16.
It should also be noted that the addition of Zn 2+ to isoD7-Aβ16 leads to its almost complete oligomerization and precipitation [9], as evident in the disappearance of the corresponding NMR signal ( Supplementary Fig. 3) However, in the presence of HAEE, Zn 2+ did not diminish the isoD7-Aβ16 signal ( Supplementary Fig. 3), highlighting the protective role of HAEE against its oligomerization.
It has been shown that an Aβ1-16 variant with the socalled English (H6R) mutation (H6R-Aβ16) exists as a mixture of monomeric and dimeric forms in the presence of Zn 2+ [9]. The dimeric form has a well-resolved characteristic signal of the V12 methyl group at 0.2 ppm, which allows for a quantitative assessment of the dimeric state. The addition of HAEE to H6R-Aβ16 in the presence of Zn 2+ leads to a disappearance of the dimeric form (Fig.  1C, Supplementary Fig. 4). Of note, the affinity of Zn 2+ to HAEE is almost an order of magnitude less than for H6R-Aβ16 ( Supplementary Fig. 5). Thus, the anti-dimerization effect of HAEE must be achieved via disrupting the H6R-Aβ16 dimerization interface, not by competing for zinc.
To assess the stability of the isoD7-Aβ16:Zn 2+ :HAEE and isoD7-Aβ42:Zn 2+ :HAEE complexes, the RMSD values were calculated as a graph showing the divergence of a structure in every MD trajectory frame from the final structure ( Supplementary Fig. 7). The final structures of complexes remained stable. As the isoD7-Aβ42:Zn 2+ :HAEE structure features 4 additional intramolecular hydrogen bonds, it less flexible than the isoD7-Aβ16:Zn 2+ :HAEE structure (Supplementary Fig. 8).
Our previous experimental studies [9] and MD modeling [27] showed that in zinc-induced aggregation of Aβ, the residues coordinating zinc in the 11 EVHH 14 site play a pivotal role. In this study, we show that the HAEE tetrapeptide efficiently binds these residues forming stable complexes, therefore these residues cannot participate in other interactions. Thus, we assume that the presence of HAEE tetrapeptide may prevent the aggregation of Aβ into unstructured conglomerates.

Exogenous HAEE prevents isoD7-Аβ42:Zn 2+ -mediated accelerated amyloidosis in the C. elegans model of AD
To study the therapeutic potential of HAEE in mitigating isoD7-Aβ42:Zn 2+ -mediated pathology in vivo, we adopted an improved C. elegans model of Aβ42 toxicity (CL2120) that has been extensively used for AD research and drug screenings [28][29][30][31][32]. As the time required for a spontaneous isomerization of Asp7 in Aβ exceeds the lifespan of nematodes, it is highly unlikely that endogenous isoD7-Aβ would occur in CL2120 animals. Exogenous peptides, including Aβ-derived peptides, were previously shown to penetrate into C. elegans tissues upon addition to the growth or incubation medium [33][34][35], giving us an opportunity to study the effects of exogenous isoD7-Aβ and zinc ions on intrinsic Aβ42 pathology. CL2120 and transgenic control (CL2122) nematodes were treated with Aβ42 or isoD7-Aβ42, with and without Zn 2+ , and amyloid aggregates visualized in live worms using amyloid-specific X-34 dye ( Fig. 2A, Supplementary  Fig. 9) [36]. In accordance with the previous data [28,37], we observed age-dependent accumulation of Aβ42 in the muscle tissue of CL2120 nematodes, but not in the CL2122 control (Supplementary Fig. 9). In worms exposed to the isoD7-Aβ42/Zn 2+ mixture, the area covered with plaques increased by 78% ( Fig. 2A and B). Remarkably, the isoD7-Aβ42/Zn 2+ -induced amyloidosis was completely negated in the presence of HAEE peptide, as both the number of plaques and plaque area reduced back to the untreated group values ( Fig. 2A and B). Notably, animals treated with isoD7-Aβ42 in the absence of zinc, or with Aβ42, with or without Zn 2+ , did not show any increase in the amyloid load (Fig. 2B, Supplementary  Fig. 10).
Thus, exogenous isoD7-Aβ42, not Aβ42, is capable of promoting Aβ42 amyloidosis in live animals in a Zn-dependent manner, whereas HAEE abrogates this pathological process.

HAEE prevents isoD7-Аβ42:Zn 2+ -induced paralysis and decelerates aging in AD model animals
The deposition of amyloid aggregates reflects the increased concentration of toxic amyloid oligomers, which can be deleterious to surrounding tissue. In CL2120 nematodes Aβ42 is expressed in body wall muscle cells, resulting in severe age progressive-paralysis [28]. Therefore, we studied if the isoD7-Aβ42/Zn 2+ mixture affects the paralysis of nematodes. We found that isoD7-Aβ42/Zn 2+ accelerates paralysis in CL2120 animals, whereas HAEE suppresses this effect (Fig. 2С). The effect was not observed for Aβ42/Zn 2+ nor amyloid peptides in the absence of Zn 2+ (Supplementary Fig. 11, Supplementary Table 2).
Next, we studied how the lifespan of nematodes is affected by exogenous amyloid peptides and zinc. CL2120 and CL2122 animals have similar lifespans at 20°C [38]. The addition of Aβ42, with or without zinc ions, to NGM did not affect the mean lifespan of CL2120 or CL2122 animals ( Supplementary Fig. 12, Supplementary  Table 3). In contrast, the addition of the isoD7-Aβ42 peptide to Zn 2+ -containing media significantly decreased the lifespan of CL2120 animals, not control CL2122 animals (Fig. 2D, Supplementary Fig. 12, Supplementary  Table 4). Without Zn 2+ , the negative effect of exogenous isoD7-Aβ42 on the Aβ42 transgenic animals was not detected.
Nematodes accumulate zinc in response to high zinc diet, and dietary zinc can be toxic to C. elegans [39,40]. Taking into account the higher affinity of isoAsp7containing peptide to zinc [10], it could promote toxicity by increasing the zinc uptake in a chelated form. However, the incubation medium contained only 20 µM of zinc, which is two orders of magnitude below the IC50 [39], thus arguing that the toxicity is due to isoD7-Aβ itself.
Strikingly, the addition of HAEE completely restored the shortened lifespan of isoD7-Aβ42 + Zn 2+ -treated CL2120 animals. HAEE itself has no effect on the lifespan of CL2120 or CL2122 animals ( Supplementary  Fig. 13, Supplementary Table 4). Thus, we conclude that systemic amyloidosis promoted by exogenous isoD7-Aβ42 in the Zn-dependent manner shortens the animals' lifespan, which can be cured by a rationally designed anti-isoD7-Aβ42 tetrapeptide (HAEE).

DISCUSSION
Alzheimer's disease has a multifactorial pathology [41][42][43][44][45]. One of the main neuromorphological signs of AD is dense and diffused extracellular aggregates of Aβ -the amyloid plaques in brain tissue [1]. Accruing evidence suggests that the pathological cascade of AD is triggered by the accumulation of soluble neurotoxic Aβ oligomers [46]. In addition to the well-known effect of zinc ions on Aβ aggregation [11,12], the formation of neurotoxic oligomers can be stimulated by the post-translational modifications of Aβ, such as Asp7 isomerization [8].
To identify the fundamental role of non-covalent complexes between Zn 2+ and isoD7-Aβ in triggering pathological aggregation of endogenous Aβ molecules, we used the HAEE tetrapeptide as a specific molecular tool capable of binding Aβ and disrupting its metaldependent dimerization interface. The formation of stable complexes of beta-amyloid peptides ( 11 EVHH 14 region) with HAEE, in the presence of Zn 2+ , is shown using SPR (Fig. 1B, Supplementary Table 1). Based on the structure of the zinc-bound H6R-Aβ16 dimer [9], models of the isoD7-Aβ complex with HAEE and Zn 2+ for the fulllength peptide and its metal-binding domain were created (Fig. 1D). Other potential Zn 2+ -interacting sites are predicted to further stabilize the complex via strong electrostatic forces, consistent with the results of the 200 ns MD run (Supplementary Fig. 7). NMR studies further show that HAEE shifts the equilibrium between monomeric and dimeric Aβ towards its monomeric form in the presence of zinc ions (Fig. 1C), indicating that it disrupts dimerization of Aβ via blocking the primary zincrecognition site at 11 EVHH 14 .
The in vivo data validates our mechanistic model of Zn 2+ -mediated amyloidosis. Strikingly, the amyloid burden in nematodes treated with isoD7-Aβ increased almost two-fold if zinc was present in the NGM medium (Fig. 2). The simultaneous presence of zinc ions and isoD7-Aβ in the diet also resulted in a significant enhancement in animal paralysis and shortening of the lifespan. If either zinc was absent from the medium or non-modified Aβ42 was used for the treatment, the changes in the amyloid load, paralysis, and lifespan were not detected.
It has been previously shown that, unlike Aβ42, exogenous isoD7-Aβ42 stimulates amyloidosis in transgenic mice, but the role of zinc in this process remained unknown. The present results imply that soluble Aβ species form zinc-bound complexes that enter C. elegans tissue. In the case of isoD7-Aβ42, such complexes promote amyloidosis and toxicity leading to animal paralysis and lifespan reduction. In the presence of HAEE, the combination of Zn 2+ and isoD7-Aβ fails to induce amyloidosis and associated pathologies in C. elegans (Fig. 2), which demonstrates that oligomeric isoAβ:Zn assemblies trigger the morbific effect. Notably, the HAEE peptide did not affect the endogenous amyloidosis in untreated transgenic C. elegans.
Taken together, the experiments with C. elegans demonstrate, for the first time, the importance of both zinc ions and the isomerization of Asp7 for systemic toxic aggregation of native Aβ. They also identify HAEE as a small molecular agent capable of blocking this pathological process at the organismal level, suggesting a novel anti-amyloid therapeutic approach.

Author Contributions
VAM, AAM and EN conceptualized this study. VAM, EPB, BP and EN designed the experiments. EPB, SE, BP, and OK performed all C. elegans experiments. VIP performed NMR experiments and analysis. AAA performed modelling and MD simulations. SAK performed SPR experiments. VAM, EPB, SE, BP, and EN analyzed data. VAM, AAM and EN wrote the paper with input from all co-authors.

Data and materials availability
The data that support the findings of this study are either included in the Manuscript and Supplementary information or available from the corresponding author upon reasonable request.