Ambient Benzo[a]pyrene’s Effect on Kinetic Modulation of Amyloid Beta Peptide Aggregation: A Tentative Association between Ultrafine Particulate Matter and Alzheimer’s Disease

Long-time exposure to ambient ultrafine particles is associated with an increased risk of neurodegenerative diseases such as Alzheimer’s disease (AD), which is triggered by the aggregation of Aβ peptide monomers into toxic oligomers. Among different ultrafine air pollutants, polycyclic aromatic hydrocarbons (PAHs) are known to have a negative neural impact; however, the impact mechanism remains obscure. We herein examined the effect of Benzo[a]Pyrene (B[a]P), one of the typical PAHs on Aβ42 oligomerization using all-atom molecular dynamics simulations. In particular, the simulations were performed using four molecules of Aβ42 in the presence of 5.00 mM, 12.5 mM, and 50.0 mM of B[a]P. The results revealed strong hydrophobic interactions between Aβ42 peptides and B[a]P, which in turn resulted in increased interpeptide electrostatic interactions. Furthermore, 5.00 mM of B[a]P accelerated the kinetics of the formation of peptide tetramer by 30%, and stabilized C-terminus in Aβ42 peptides, suggesting consequent progression of AD in the presence of 5.00 mM B[a]P. In contrast, 12.5 mM and 50.0 mM of B[a]P decreased interpeptide interactions and H-bonding due to the aggregation of numerous B[a]P clusters with the peptides, suppressing oligomerization kinetics of Aβ42 peptides by 13% and 167%, respectively. While the study elucidates the effect of small environmental hydrophobic molecules on the formation of Aβ oligomers, the impact of ambient ultrafine particles on AD in the complex composition of the environmental realm requires further systematic delving into the field.


Introduction
Environmental air pollutants are known to have a negative neural impact [1], with the effect ranging from an increased risk of autism spectrum disorders, brain volumetric changes, brain inflammation, cerebrovascular and neuropsychiatric disorders, and the hallmarks of Alzheimer's disease (AD) [2][3][4][5][6]. The concern about the development of AD-like neurodegenerative diseases increases with the growth of the elderly population globally. AD is associated with the development of amyloid plaques due to the aggregation of amyloid beta (Aβ) peptide monomers into soluble oligomers, and the formation of neurofibrillary tangles in the human brain [7]. The pathogenesis of AD also implicates genetic and environmental factors [8].
Among different air pollutants, Polycyclic Aromatic Hydrocarbons (PAHs) are associated with an increased risk for neurodegeneration [9]. Benzo[a]pyrene (B[a]P, C 20 H 12 ) is a typical PAH, with five aromatic rings in its structure. B[a]P is usually generated from incomplete combustion of organic material, motor-vehicle exhaust, and from cooking and smoking. According to source apportionment measurements, the average concentration the 1:1 ratio between the peptide and air pollutant, as was used in our previous study, where we simulated systems with 1 monomer and 1 B[a]P molecule [26]. The highest concentration of B[a]P used in the simulations was 50 mM, with the 10:1 ratio between B[a]P and peptides, as was previously used in the experimental study performed by Wallin et al. [27]. The choice of 12.5 mM was taken as an intermediate concentration between the two values. Considering that the simulation box is a simple model of a physical system, the aggregation of peptides occurs faster in the MD simulations, while the results of the MD studies represent the general trend that would be expected to be found in an actual system.

Methodology
Atomistic molecular dynamics simulations were performed via Gromacs 2019.6 software [31]. Gromos54a7 forcefield parameters were chosen based on the literature validations [32] and our previous study with the validation of the density of B[a]P at 20 • C [26]. The molecular dynamics simulations were performed in a simulation box with the dimensions of 11 × 11 × 11 nm 3 . Four Aβ 42 monomers (PDB ID: 1Z0Q, with a total charge of −2 on each peptide) were inserted in a simulation box, keeping the concentration of peptides at 5.00 mM. The system was solvated via SPC water model. 0.15 M of NaCl salt was added as a buffer environment. The topology parameters and optimized geometry of B[a]P molecule (C 20 H 12 ) were taken from the Automated Topology Builder (ATB, Version 3.0) [33]. The concentration of B[a]P varied from 0 mM and 5.00 mM, 12.5 mM, and 50.0 mM, corresponding to 4, 10, and 40 molecules of B[a]P. Due to the limitations of the simulation time and box size, the concentrations of Aβ 42 peptide and B[a]P molecules in the simulated systems were comparatively higher than the molecular concentrations normally found in the human blood [15]. However, the relative amount of B[a]P to Aβ, which is 10:1, is similar to those used in the in vitro experiments [27]. The number of molecules used in the simulated systems is shown in Table 1. Energy minimization was performed with the steepest descent algorithm, applying periodic boundary conditions in all directions, and setting the maximum force for convergence of 100 kJ mol −1 nm −1 . After that, NVT-equilibration step with H-bonds constraints was performed for 25 ps with a time step of 0.5 fs. Next, the NPT-equilibration step with all-bonds constraints was performed for 100 ps with a time step of 2 fs, at reference pressure of 1 bar. LINCS (LINear Constraint Solver) constraint algorithm [34] with LINCS iterations of 1 was applied at reference temperature of 298 K. Short-range cutoff distance of 1 nm was applied for both electrostatics and van der Waals interactions with Verlet cut-off scheme algorithm. PME (Particle-mesh Ewald) was set for long-range electrostatic interactions. Molecular dynamics simulations were performed for 500 ns with a time step of 2 fs, considering reaching an equilibrium within a simulated time in all systems under the study, as discussed below. The output coordinates, velocities, and energies were saved every 4 ns.
The oligomerization (and kinetics) of four Aβ 42 peptides were investigated by using cluster and intermolecular distance analyses. The intermolecular distances were calculated based on the center of masses (COM) of the residues and taking the average values between the distances between COM of peptides 1 and 2, peptides 1 and 3, peptides 1 and 4, etc. The formation of the clusters was studied for the defined groups of the residues, "four peptides" and "peptides and B[a]P", to analyze the formation of the interpeptide and peptide-B[a]P clusters, respectively. The cluster analyses were performed setting 0.35 nm Toxics 2022, 10, 786 4 of 13 as a maximum distance parameter between the center of masses of the residues within the defined group to be defined as a cluster. Time-evolution of solvent accessible surface area (SASA), radius of gyration (RoG), and secondary structure analyses were performed to investigate the deviations in the structure of the peptides within 500 ns of the simulations. The (radial distribution function) rdf analysis was performed to investigate the probability of finding "peptides" and "B[a]P" residues from "peptides" residues using the COM of the residues. H-bonds analyses, root-mean square fluctuations (RMSF), and (rdf) analyses were performed for the last 30 ns of the simulations to investigate the structure of peptides and intermolecular interactions in the end of the simulations. In addition, visual molecular dynamics (VMD) software [35] was used to visualize the systems under the study. Finally, the non-bonded intermolecular interactions were studied for the last 10 ns of the MD simulations. In particular, the short-range (SR) interactions with the cut-off distance of 1 nm, and long-range (LR) interaction energies were calculated separately. In addition, the Lennard-Jones (LJ) potential and Coulombic (Coul) potential energies were quantified, indicating the repulsion-dispersion and electrostatics interactions, respectively.
Finally, an additional short replica of each system was simulated independently for 250 ns, starting from the randomly inserted molecules in the simulation box. The results of the simulations were validated via interpeptide distance and SASA analyses.

Results and Discussion
The kinetics of the tetramerization of Aβ 42 peptides was initially studied in the absence of B[a]P and in the presence of 4 B[a]P molecules via interpeptide distance analysis ( Figure 1A). Similarly, the effect of B[a]P concentration on the oligomerization was investigated by interpeptide distance analysis ( Figure 1B), followed by the peptide-B[a]P distance analysis ( Figure 1C).
Toxics 2022, 10, x FOR PEER REVIEW 4 of 13 4, etc. The formation of the clusters was studied for the defined groups of the residues, "four peptides" and "peptides and B[a]P", to analyze the formation of the interpeptide and peptide-B[a]P clusters, respectively. The cluster analyses were performed setting 0.35 nm as a maximum distance parameter between the center of masses of the residues within the defined group to be defined as a cluster. Time-evolution of solvent accessible surface area (SASA), radius of gyration (RoG), and secondary structure analyses were performed to investigate the deviations in the structure of the peptides within 500 ns of the simulations. The (radial distribution function) rdf analysis was performed to investigate the probability of finding "peptides" and "B[a]P" residues from "peptides" residues using the COM of the residues. H-bonds analyses, root-mean square fluctuations (RMSF), and (rdf) analyses were performed for the last 30 ns of the simulations to investigate the structure of peptides and intermolecular interactions in the end of the simulations. In addition, visual molecular dynamics (VMD) software [35] was used to visualize the systems under the study. Finally, the non-bonded intermolecular interactions were studied for the last 10 ns of the MD simulations. In particular, the short-range (SR) interactions with the cut-off distance of 1 nm, and long-range (LR) interaction energies were calculated separately. In addition, the Lennard-Jones (LJ) potential and Coulombic (Coul) potential energies were quantified, indicating the repulsion-dispersion and electrostatics interactions, respectively. Finally, an additional short replica of each system was simulated independently for 250 ns, starting from the randomly inserted molecules in the simulation box. The results of the simulations were validated via interpeptide distance and SASA analyses.

Results and Discussion
The kinetics of the tetramerization of Aβ42 peptides was initially studied in the absence of B[a]P and in the presence of 4 B[a]P molecules via interpeptide distance analysis ( Figure 1A). Similarly, the effect of B[a]P concentration on the oligomerization was investigated by interpeptide distance analysis ( Figure 1B), followed by the peptide-B[a]P distance analysis ( Figure 1C).  The analysis of the interpeptide distances showed that in the absence of B[a]P molecules, four Aβ 42 peptides aggregated within the simulated time; in particular, within 100 ns of the simulation ( Figure 1A). A different trend was observed by Brown et al. [36], wherein the tetramerization of Aβ 42 was seen within 250 ns of the simulation. The difference in the tetramerization time could be the usage of older forcefield parameters (GROMOS96 53A6), higher temperature of simulations (310 K), and large simulation box (with dimensions of 12.7 nm) by Brown and Bevan [36]. According to Figure 1A, the presence of 4 B[a]P molecules significantly decreased the time required for the oligomerization. The peptides aggregated within 50 ns of the simulation, which was the fastest aggregation among the systems under the study ( Figure 1B). The results obtained from the interpeptide distance analysis were validated via an additional short replica for 250 ns of each system. Additional plots for each system under the study are available in the Supplementary Material ( Figure S1). While quantitatively the values were different, qualitatively the trends remained the same.
With increasing the concentration of B[a]P molecules, i.e., using 10 and 40 molecules, the time required for tetramerization increased, and became 100 ns and 200 ns for the two systems, respectively. In addition, according to Figure 1C, the distance analyses between peptides and B[a]P molecules showed that B[a]P molecules were also bound to the peptides within 70 ns of the simulations in all systems under the study, indicating the formation of agglomerate encompassing the PAHs and the peptides. Furthermore, the aggregation kinetics of four Aβ 42 peptides was analyzed in terms of the growth of the interpeptide clusters (Figure 2A), as well as the formation of the clusters of B[a]P and peptides ( Figure 2B).
Toxics 2022, 10, x FOR PEER REVIEW 5 of 13 Figure 1. Time-evolution of (A,B) average interpeptide distances during 500 ns of the simulation, (C) average distances between Aβ42 peptides and B[a]P molecules during 500 ns of the simulation.
The analysis of the interpeptide distances showed that in the absence of B[a]P molecules, four Aβ42 peptides aggregated within the simulated time; in particular, within 100 ns of the simulation ( Figure 1A). A different trend was observed by Brown et al. [36], wherein the tetramerization of Aβ42 was seen within 250 ns of the simulation. The difference in the tetramerization time could be the usage of older forcefield parameters (GRO-MOS96 53A6), higher temperature of simulations (310 K), and large simulation box (with dimensions of 12.7 nm) by Brown and Bevan [36]. According to Figure 1A, the presence of 4 B[a]P molecules significantly decreased the time required for the oligomerization. The peptides aggregated within 50 ns of the simulation, which was the fastest aggregation among the systems under the study ( Figure 1B). The results obtained from the interpeptide distance analysis were validated via an additional short replica for 250 ns of each system. Additional plots for each system under the study are available in the Supplementary material ( Figure S1). While quantitatively the values were different, qualitatively the trends remained the same.
With increasing the concentration of B[a]P molecules, i.e., using 10 and 40 molecules, the time required for tetramerization increased, and became 100 ns and 200 ns for the two systems, respectively. In addition, according to Figure 1C, the distance analyses between peptides and B[a]P molecules showed that B[a]P molecules were also bound to the peptides within 70 ns of the simulations in all systems under the study, indicating the formation of agglomerate encompassing the PAHs and the peptides. Furthermore, the aggregation kinetics of four Aβ42 peptides was analyzed in terms of the growth of the interpeptide clusters (Figure 2A), as well as the formation of the clusters of B[a]P and peptides ( Figure 2B). According to Figure 2A, the rate of tetramerization of four peptides was fastest in the presence of 4 B[a]P molecules (in ~52 ns of the MD run). In comparison, in the absence of B[a]P molecules, a stable single cluster of four peptides was produced in ~75 ns. In the presence of 10 B[a]P molecules, the cluster was formed in ~85 ns of the simulation, while in the presence of 40 B[a]P, formation of a stable cluster of four peptides was completed in ~200 ns of the production run. According to Figure 2B, the fastest formation of a stable peptide-B[a]P cluster occurred in ~40 ns of the simulation in the presence of 4 B[a]P mol- According to Figure 2A, the rate of tetramerization of four peptides was fastest in the presence of 4 B[a]P molecules (in~52 ns of the MD run). In comparison, in the absence of B[a]P molecules, a stable single cluster of four peptides was produced in~75 ns. In the presence of 10 B[a]P molecules, the cluster was formed in~85 ns of the simulation, while in the presence of 40 B[a]P, formation of a stable cluster of four peptides was completed in~200 ns of the production run. According to Figure 2B Overall, according to cluster analyses, the presence of 5.00 mM B[a]P enhanced the oligomerization kinetics, based on the time required to make one stable cluster of four Aβ 42  [37], rather than the intrinsic interpeptide hydrophobic interactions [38]. The secondary structure of the interpeptide clusters was further analyzed for the last 30 ns of the simulations (Figure 3), wherein the tetramerized, clustered structure was stable. In addition, the intermolecular clusters observed at the end of the simulations, along with the initial monomer structure, were visualized via VMD (Figure 4).
ecules. In comparison, additional time was required for the complete aggregation of peptides and B[a]P molecules in the systems with 10 B[a]P molecules (~85 ns of the simulation) and 40 B[a]P molecules (~155 ns of the simulation).
Overall, according to cluster analyses, the presence of 5.00 mM B[a]P enhanced the oligomerization kinetics, based on the time required to make one stable cluster of four Aβ42 peptide monomers, by 30%, in comparison to the aggregation kinetics of peptides in the absence of B[a]P. In contrast, the presence of 12.5 mM B[a]P and 50.0 mM B[a]P decreased the aggregation kinetics of peptides by 13% and 167%, respectively. In addition, in the system with 10 B[a]P molecules, the formation of interpeptide clusters and formation of peptide-B[a]P clusters occurred simultaneously, while in the systems with 4 and 40 B[a]P molecules, the formation of a stable interpeptide cluster occurred only after all B[a]P molecules were bound to peptides. The results indicated that oligomerization was essentially driven by the hydrophobic interactions between B[a]P and Aβ42 peptides [37], rather than the intrinsic interpeptide hydrophobic interactions [38]. The secondary structure of the interpeptide clusters was further analyzed for the last 30 ns of the simulations (Figure 3), wherein the tetramerized, clustered structure was stable. In addition, the intermolecular clusters observed at the end of the simulations, along with the initial monomer structure, were visualized via VMD (Figure 4).  ecules. In comparison, additional time was required for the complete aggregation of peptides and B[a]P molecules in the systems with 10 B[a]P molecules (~85 ns of the simulation) and 40 B[a]P molecules (~155 ns of the simulation).
Overall, according to cluster analyses, the presence of 5.00 mM B[a]P enhanced the oligomerization kinetics, based on the time required to make one stable cluster of four Aβ42 peptide monomers, by 30%, in comparison to the aggregation kinetics of peptides in the absence of B[a]P. In contrast, the presence of 12.5 mM B[a]P and 50.0 mM B[a]P decreased the aggregation kinetics of peptides by 13% and 167%, respectively. In addition, in the system with 10 B[a]P molecules, the formation of interpeptide clusters and formation of peptide-B[a]P clusters occurred simultaneously, while in the systems with 4 and 40 B[a]P molecules, the formation of a stable interpeptide cluster occurred only after all B[a]P molecules were bound to peptides. The results indicated that oligomerization was essentially driven by the hydrophobic interactions between B[a]P and Aβ42 peptides [37], rather than the intrinsic interpeptide hydrophobic interactions [38]. The secondary structure of the interpeptide clusters was further analyzed for the last 30 ns of the simulations (Figure 3), wherein the tetramerized, clustered structure was stable. In addition, the intermolecular clusters observed at the end of the simulations, along with the initial monomer structure, were visualized via VMD (Figure 4).  The results of the secondary structure analysis obtained from our previous study [26] showed that in the presence of 1 B[a]P molecule, high amounts of the coils, bends, and β-content with low amount of helices were observed in the secondary structure of the peptide monomer. The results of this study (Figure 3) showed that the secondary structure of the oligomers would also depend on the concentration of the B[a]P molecules present in the simulation box. In particular, the analysis of the time-averaged secondary structure of Aβ 42 peptides showed that in the presence of 4 B[a]P molecules, the coil region was predominant (38%). In comparison, in the presence of 10 and 40 B[a]P molecules, the helix region was prevalent (34% and 36%, respectively). Typically, the progression of Alzheimer's disease is associated with the formation of β-sheet fibrils [39,40], however, more simulation time will be required for the formation of the stable β-sheets from the random coils in the peptide oligomers [41]. Interestingly, the β-content did not significantly vary with different B[a]P concentrations, with 6-9% of the β-content observed in all systems under the study. Nonetheless, the formation of the compact coil structures in Aβ oligomers is also an important step toward the formation of β-sheets [42]. In particular, in the presence of 4 B[a]P molecules, the high number of coils in the secondary structure of the peptides was correlated with enhanced oligomerization kinetics.
According to Figure Figure 4E). These observations showed that β-sheets were mainly formed in the regions of C-terminus, considered as a possible nucleation site for the self-aggregation, as was highlighted in the earlier literature [43,44]. In addition, in the presence of 4 B[a]P, the tetramerization occurred with the formation of oligomers with increased coil region, that could be expected to form β-sheets further [45]. In comparison, in the systems with 10 and 40 B[a]P molecules, the binding of numerous B[a]P to peptides via aromatic π-π interactions [46] resulted in the formation of B[a]P-peptide agglomerates. Consequently, typical oligomerization was suppressed due to the presence of numerous B[a]P molecules and their clusters, resulted in steric hindrance [47], as shown in Figure 4D,E. To investigate the deviations in the peptide structure with the formation of intermolecular clusters, RMSF, RoG, SASA, and H-bonds analyses were further performed ( Figure 5, Table 2).      1 nm), indicating a similar effect of the tetramer stabilization. Moreover, in this system under study, the enhanced fluctuations were observed in the N-and Cterminuses, which were correlated with the suppression of oligomerization (RMSF values up to~0.23 nm for Aβ 1-2 , and~0.14 nm for Aβ [38][39][40][41][42]. Similarly, enhanced fluctuations in the positions of the amino acids of the N-terminus (Aβ 1-2 , RMSF values of~0.28 nm), and C-terminus (Aβ 37-41 segment RMSF values of~0.14-0.21 nm) were observed in the system with 40 B[a]P molecules in the end of the simulation, indicating increased movement of the aminoacids in the peptide terminuses due to the interactions with large amounts of B[a]P molecules and the hydrophobic interactions with the PAHs, as was discussed previously.
This observation was further corroborated with the time-evolution of the radius of gyration (RoG) of the peptide. The RoG values, averaged between four Aβ 42 peptides, decreased from the initial value of 1.6 nm to~1.1 nm within the simulation time in the systems with no B[a]P and in the presence of 4 B[a]P molecules ( Figure 5B). In contrast, in the presence of 10 B[a]P and 40 B[a]P molecules, the radius of gyration deviated more significantly, up to~1.3 nm and~1.4 nm, respectively, due to the interactions with large amounts of B[a]P molecules.
In agreement with cluster and intermolecular distance analyses, for the systems with no B[a]P molecules, with 4 B[a]P, and 10 B[a]P molecules, the SASA values significantly decreased within first 100 ns of the MD simulations, indicating the interpeptide binding with consequent loss of peptide surface area available for solvent ( Figure 5C). In addition, according to Figure 5C and Table 2 (Table 2). Overall, RoG, SASA, and H-bonds analyses showed that the presence of 10 and 40 B[a]P molecules could interfere with the oligomerization process by binding to Aβ 42 peptides, leading to elevated radius of gyration, SASA of Aβ 42 peptides, and suppressed interpeptide H-bonding.
Furthermore, the radial distribution function (rdf) analysis showed that high interpeptide interactions were observed in the systems with no B[a]P molecules (maximum peaks of~55 at 0.5 nm and~53 at 0.6 nm on Figure 6A), and in the presence of 4 B[a]P molecules (maximum peak of~53 at 0.6 nm on Figure 6A). In comparison, decreased interpeptide interactions in the presence of 10 B[a]P molecules (maximum peak value of 50 at 0.6 nm on Figure 6A), and in the presence of 40 B[a]P molecules (highest peaks of~45 at 0.4 nm and 0.6 nm on Figure 6A), indicated that interpeptide interactions were decreased with the addition of higher amounts of B[a]P molecules. According to the rdf analysis of peptide-B[a]P interactions ( Figure 6B), strong peptide-B[a]P interactions were observed in the system with 4 B[a]P molecules (maximum peaks of~30 at 0.47 nm and~60 at 0.72 nm on Figure 6B). In comparison, low peptide-B[a]P interactions were observed in the systems with 10 B[a]P molecules (maximum peaks of~17 at 0.47 nm and~30 at 0.72 nm on Figure 6B) and 40 B[a]P molecules (maximum peaks of~10 at 0.4 nm and~20 at 0.76 nm on Figure 6B).
in the systems with 10 B[a]P molecules (maximum peaks of ~17 at 0.47 nm and ~30 at 0.72 nm on Figure 6B) and 40 B[a]P molecules (maximum peaks of ~10 at 0.4 nm and ~20 at 0.76 nm on Figure 6B).  i.e., 4 molecules, accelerated the formation of Aβ42 peptide tetramers by 30% and stabilized C-terminus of peptides, suggesting consequent progression of Alzheimer's disease. Nonetheless, the effect of ultrafine air pollutants on AD would be more complicated due to the more complex composition of pollutants present in the environment. The energy analysis was further performed for the last 10 ns of the simulations to investigate the type of molecular interactions. According to Table 3, in the presence of B[a]P molecules, short-range electrostatic interpeptide interactions increased (Coul-SR), while short-range Lennard-Jones interactions (LJ-SR) between peptides decreased. These observations were also correlated to the enhanced hydrophobic interactions between peptides and B[a]P (LJ-SR), related to the aggregation of peptides and B[a]P molecules. Table 3. Short-range (SR) and long-range (LR) Coulombic and Lennard-Jones potential between peptides, and peptide-B[a]P, observed in the last 10 ns of the simulations in the systems under the study.

System
Peptide

Conclusions
To conclude, molecular dynamics simulations revealed that with the addition of B[a]P molecules, electrostatic interactions between Aβ 42 peptides increased due to the enhanced B[a]P-peptide hydrophobic interactions and binding of Aβ 42 peptides and B[a]P. Although the presence of 4 B[a]P molecules did not significantly affect interpeptide H-bonding and interpeptide interactions, the presence of 10 and 40 B[a]P molecules suppressed H-bonding and decreased interpeptide interactions, observed in the end of the simulations. Overall, according to the results of our study, the presence of 5.00 mM B[a]P, i.e., 4 molecules, accelerated the formation of Aβ 42 peptide tetramers by 30% and stabilized C-terminus of peptides, suggesting consequent progression of Alzheimer's disease. Nonetheless, the effect of ultrafine air pollutants on AD would be more complicated due to the more complex composition of pollutants present in the environment.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/toxics10120786/s1, Figure S1: Time-evolution of average interpeptide distances in the systems with additional replicas; Figure S2 Data Availability Statement: The datasets generated and analyzed during the current study are available from the corresponding author on a reasonable request.

Conflicts of Interest:
The authors have no relevant financial or non-financial interest to disclose.