Changes in ficin specificity by different substrate proteins promoted by enzyme immobilization.

Ficin extract has been immobilized using different supports: glyoxyl and Aspartic/1,6 hexamethylenediamine (Asp/HA) agarose beads. The latter was later submitted to glutaraldehyde modification to get covalent immobilization. The activities of these 3 kinds of biocatalysts were compared utilizing 4 different substrates, casein, hemoglobin and bovine serum albumin and benzoyl-arginine-p-nitroanilide at pH 7 and 5. Using glyoxyl-agarose, the effect of enzyme-support reaction time on the activity versus the four substrates at both pH values was studied. Reaction time has been shown to distort the enzyme due to an increase in the number of covalent support-enzyme bonds. Surprisingly, for all the substrates and conditions the prolongation of the enzyme-support reaction did not imply a decrease in enzyme activity. Using the Asp/HA supports (with different amount of HA) differences in the effect on enzyme activity versus the different substrates are much more significant, while with some substrates the immobilization produced a decrease in enzyme activity, with in other cases the activity increased. These different effects are even increased after glutaraldehyde treatment. That way, the conformational changes induced by the biocatalyst immobilization or the chemical modification fully altered the enzyme protein specificity. This may also have some implications when following enzyme inactivation.

Tailoring the specificity of ficin versus large hemoglobin and small casein by co-immobilizing inert proteins on the immobilized enzyme layer and further modification with aldehyde dextran.

Ficin has been immobilized at full loading on glyoxyl agarose beads. Then, ficin was blocked with 2,2'-dipyridyldisulfide. To be effective, the modification must be performed in the presence of 0.5 M urea, as the enzyme was not inhibited under standard conditions, very likely because the catalytic Cys was not fully exposed to the medium. Activity could be fully recovered by incubation with 1 M mercaptoethanol. This biocatalyst could hydrolyze hemoglobin and casein. The objective of this paper was to increase the enzyme specificity versus small proteins by generating steric hindrances to the access of large proteins. The step by step blocking via ionic exchange of the biocatalyst with aminated bovine serum albumin (BSA), aldehyde dextran and a second layer of aminated BSA produced a biocatalyst that maintained its activity versus small synthetic substrates, increased the biocatalyst stability, while reduced its activity to over 50 % versus casein. Interestingly, this treatment almost fully annulled the activity versus hemoglobin, more effectively at 37 °C than at 55 °C. The biocatalyst could be reused 5 times without changes in activity. The changes could be caused by steric hindrances, but it cannot be discarded some changes in enzyme sequence specificity caused by the modifications.

Support Enzyme Loading Influences the Effect of Aldehyde Dextran Modification on the Specificity of Immobilized Ficin for Large Proteins.

It has been reported that the modification of immobilized glyoxyl-ficin with aldehyde dextran can promote steric hindrances that greatly reduce the activity of the immobilized protease against hemoglobin, while the protease still maintained a reasonable level of activity against casein. In this paper, we studied if this effect may be different depending on the amount of ficin loaded on the support. For this purpose, both the moderately loaded and the overloaded glyoxyl-ficin biocatalysts were prepared and modified with aldehyde dextran. While the moderately loaded biocatalyst had a significantly reduced activity, mainly against hemoglobin, the activity of the overloaded biocatalyst was almost maintained. This suggests that aldehyde dextran was able to modify areas of the moderately loaded enzyme that were not available when the enzyme was overloaded. This modification promoted a significant increase in biocatalyst stability for both biocatalysts, but the stability was higher for the overloaded biocatalyst (perhaps due to a combination of inter- and intramolecular crosslinking).

Tuning almond lipase features by the buffer used during immobilization: The apparent biocatalysts stability depends on the immobilization and inactivation buffers and the substrate utilized.

The lipase from Prunus dulcis almonds was inactivated under different conditions. At pH 5 and 9, enzyme stability remained similar under the different studied buffers. However, when the inactivation was performed at pH 7, there were some clear differences on enzyme stability depending on the buffer used. The enzyme was more stable in Gly than when Tris was employed for inactivation. Then, the enzyme was immobilized on methacrylate beads coated with octadecyl groups at pH 7 in the presence of Gly, Tris, phosphate and HEPES. Its activity was assayed versus triacetin and S-methyl mandelate. The biocatalyst prepared in phosphate was more active versus S-methyl mandelate, while the other ones were more active versus triacetin. The immobilized enzyme stability at pH 7 depends on the buffer used for enzyme immobilization. The buffer used in the inactivation and the substrate used determined the activity. For example, glycine was the buffer that promoted the lowest or the highest stabilities depending on the substrate used to quantify the activities.

Catalytic mechanism and kinetics of malate dehydrogenase.

Malate dehydrogenase (MDH) is a ubiquitous and central enzyme in cellular metabolism, found in all kingdoms of life, where it plays vital roles in the cytoplasm and various organelles. It catalyzes the reversible NAD+-dependent reduction of L-malate to oxaloacetate. This review describes the reaction mechanism for MDH and the effects of mutations in and around the active site on catalytic activity and substrate specificity, with a particular focus on the loop that encloses the active site after the substrates have bound. While MDH exhibits selectivity for its preferred substrates, mutations can alter the specificity of MDH for each cosubstrate. The kinetic characteristics and similarities of a variety of MDH isozymes are summarized, and they illustrate that the KM values are consistent with the relative concentrations of the substrates in cells. As a result of its existence in different cellular environments, MDH properties vary, making it an attractive model enzyme for studying enzyme activity and structure under different conditions.

Flexible active-site loops fine-tune substrate specificity of hyperthermophilic metallo-oxidases.

Hyperthermophilic ('superheat-loving') archaea found in high-temperature environments such as Pyrobaculum aerophilum contain multicopper oxidases (MCOs) with remarkable efficiency for oxidizing cuprous and ferrous ions. In this work, directed evolution was used to expand the substrate specificity of P. aerophilum McoP for organic substrates. Six rounds of error-prone PCR and DNA shuffling followed by high-throughput screening lead to the identification of a hit variant with a 220-fold increased efficiency (kcat/Km) than the wild-type for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) without compromising its intrinsic activity for metal ions. The analysis of the X-ray crystal structure reveals four proximal mutations close to the T1Cu active site. One of these mutations is within the 23-residues loop that occludes this site, a distinctive feature of prokaryotic MCOs. The increased flexibility of this loop results in an enlarged tunnel and one additional pocket that facilitates bulky substrate-enzyme interactions. These findings underscore the synergy between mutations that modulate the dynamics of the active-site loop enabling enhanced catalytic function. This study highlights the potential of targeting loops close to the T1Cu for engineering improvements suitable for biotechnological applications.

Enzymatic debittering of cheese flavoring and bitterness characterization of peptide mixture using sensory and peptidomics approach.

Peptides in cheese flavoring produced through proteolysis plus fermentation generated bitterness. Bitterness of individual peptide can be quantified using quantitative structure-activity relationship, where molecular mass (M), hydrophobicity, residues, C-terminal hydrophobic amino acids (C-HAAs), and N-terminal basic ones (N-BAAs) are crucial. However, their accumulative influence on the overall bitterness of peptide mixture remains unknown. This study delved into extensive proteolysis to debitter and to correlate the multi-influencing factors of peptides and the collective bitterness. As hydrolysis increased from 7.5 % to 28.0 %, bitterness reduced from 5.0 to 0.3-2.7 scores, contingent on proteases used, in which FU was optimal. The overall bitterness cannot be predicted through the summation of individual peptide bitterness, which depended on M (0.5-3 kDa) and 5-23 residues, followed by N-BAAs and C-HAAs. Analysis of enzymatic cleavage sites and substrate characteristics revealed, to more effectively debitter bovine milk protein hydrolysates, proteases specifically cleaving Pro, Leu, Phe, and Val were desired.

CryoEM analysis of the essential native UDP-glucose pyrophosphorylase from Aspergillus nidulans reveals key conformations for activity regulation and function.

Invasive aspergillosis is one of the most serious clinical invasive fungal infections, resulting in a high case fatality rate among immunocompromised patients. The disease is caused by saprophytic molds in the genus Aspergillus, including Aspergillus fumigatus, the most significant pathogenic species. The fungal cell wall, an essential structure mainly composed of glucan, chitin, galactomannan, and galactosaminogalactan, represents an important target for the development of antifungal drugs. UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) is a central enzyme in the metabolism of carbohydrates that catalyzes the biosynthesis of UDP-glucose, a key precursor of fungal cell wall polysaccharides. Here, we demonstrate that the function of UGP is vital for Aspergillus nidulans (AnUGP). To understand the molecular basis of AnUGP function, we describe a cryoEM structure (global resolution of 3.5 Å for the locally refined subunit and 4 Å for the octameric complex) of a native AnUGP. The structure reveals an octameric architecture with each subunit comprising an N-terminal α-helical domain, a central catalytic glycosyltransferase A-like (GT-A-like) domain, and a C-terminal (CT) left-handed ß-helix oligomerization domain. AnUGP displays unprecedented conformational variability between the CT oligomerization domain and the central GT-A-like catalytic domain. In combination with activity measurements and bioinformatics analysis, we unveil the molecular mechanism of substrate recognition and specificity for AnUGP. Altogether, our study not only contributes to understanding the molecular mechanism of catalysis/regulation of an important class of enzymes but also provides the genetic, biochemical, and structural groundwork for the future exploitation of UGP as a potential antifungal target. IMPORTANCE Fungi cause diverse diseases in humans, ranging from allergic syndromes to life-threatening invasive diseases, together affecting more than a billion people worldwide. Increasing drug resistance in Aspergillus species represents an emerging global health threat, making the design of antifungals with novel mechanisms of action a worldwide priority. The cryoEM structure of UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) from the filamentous fungus Aspergillus nidulans reveals an octameric architecture displaying unprecedented conformational variability between the C-terminal oligomerization domain and the central glycosyltransferase A-like catalytic domain in the individual protomers. While the active site and oligomerization interfaces are more highly conserved, these dynamic interfaces include motifs restricted to specific clades of filamentous fungi. Functional study of these motifs could lead to the definition of new targets for antifungals inhibiting UGP activity and, thus, the architecture of the cell wall of filamentous fungal pathogens.

Design and interpretation of experiments to establish enzyme pathway and define the role of conformational changes in enzyme specificity.

We describe the experimental methods and analysis to define the role of enzyme conformational changes in specificity based on published studies using DNA polymerases as an ideal model system. Rather than give details of how to perform transient-state and single-turnover kinetic experiments, we focus on the rationale of the experimental design and interpretation. We show how initial experiments to measure kcat and kcat/Km can accurately quantify specificity but do not define its underlying mechanistic basis. We describe methods to fluorescently label enzymes to monitor conformational changes and to correlate fluorescence signals with rapid-chemical-quench flow assays to define the steps in the pathway. Measurements of the rate of product release and of the kinetics of the reverse reaction complete the kinetic and thermodynamic description of the full reaction pathway. This analysis showed that the substrate-induced change in enzyme structure from an open to a closed state was much faster than rate-limiting chemical bond formation. However, because the reverse of the conformational change was much slower than chemistry, specificity is governed solely by the product of the binding constant for the initial weak substrate binding and the rate constant for the conformational change (kcat/Km=K1k2) so that the specificity constant does not include kcat. The enzyme conformational change leads to a closed complex in which the substrate is bound tightly and is committed to the forward reaction. In contrast, an incorrect substrate is bound weakly, and the rate of chemistry is slow, so the mismatch is released from the enzyme rapidly. Thus, the substrate-induced-fit is the major determinant of specificity. The methods outlined here should be applicable to other enzyme systems.

[Mechanisms of the Specificity of the CRISPR/Cas9 System in Genome Editing].

The CRISPR/Cas9 system, which was discovered recently, utilizes nucleases targeted by sequence complementarity and is originally intended to protect bacteria from foreign genetic elements. The system provided a convenient tool for manipulating the genomes of living cells. The CRISPR/Cas9 genomic editing technology moved beyond the laboratory and already found application in biotechnology and agriculture. However, off-target activity of the CRISPR/Cas9 system can cause oncogenic mutations and thus limits its use for genome editing in human cells for medical purposes. Many studies are therefore aimed at developing variants of the CRISPR/Cas9 system with improved accuracy. The review considers the mechanisms of precise and erroneous actions of Cas9 RNA-guided nuclease, natural and artificial variants of RNA-targeted nucleases, possibilities to modulate their specificity through guide RNA modifications, and other approaches to increasing the accuracy of the CRISPR/Cas9 system in genome editing.

Immobilization of Lipase B from Candida antarctica in Octyl-Vinyl Sulfone Agarose: Effect of the Enzyme-Support Interactions on Enzyme Activity, Specificity, Structure and Inactivation Pathway.

Lipase B from Candida antarctica was immobilized on heterofunctional support octyl agarose activated with vinyl sulfone to prevent enzyme release under drastic conditions. Covalent attachment was established, but the blocking step using hexylamine, ethylenediamine or the amino acids glycine (Gly) and aspartic acid (Asp) altered the results. The activities were lower than those observed using the octyl biocatalyst, except when using ethylenediamine as blocking reagent and p-nitrophenol butyrate (pNPB) as substrate. The enzyme stability increased using these new biocatalysts at pH 7 and 9 using all blocking agents (much more significantly at pH 9), while it decreased at pH 5 except when using Gly as blocking agent. The stress inactivation of the biocatalysts decreased the enzyme activity versus three different substrates (pNPB, S-methyl mandelate and triacetin) in a relatively similar fashion. The tryptophane (Trp) fluorescence spectra were different for the biocatalysts, suggesting different enzyme conformations. However, the fluorescence spectra changes during the inactivation were not too different except for the biocatalyst blocked with Asp, suggesting that, except for this biocatalyst, the inactivation pathways may not be so different.

The immobilization protocol greatly alters the effects of metal phosphate modification on the activity/stability of immobilized lipases.

Mineralization of immobilized enzymes has showed to couple the advantages of both processes. Here, the influence of the immobilization protocol on the effects of mineralization has been investigated. The lipases from Thermomyces lanuginosus and Candida rugosa were immobilized on octyl-, vinyl sulfone (VS) octyl (blocked with different nucleophiles) and glutaraldehyde- (at different pH values) agarose beads. The stability, activity and specificity of the biocatalysts were very different, both the differently blocked VS-biocatalysts and the glutaraldehyde biocatalysts prepared at different pH. All biocatalysts were submitted to mineralization using different metals. The activity, specificity and stability effects of the mineralization strongly depended on the enzyme and on the immobilization protocol. For the same enzyme, a mineralization protocol could be negative, positive or present no effect depending on the enzyme immobilization procedure and substrate. In the best cases, activity could be increased by a two-fold factor, while stability was significantly improved in many instances. These results highlight the great potential of mineralization of immobilized enzymes to improve their properties, as well as the great interactions that immobilization protocol and mineralization can exhibit. The combination of both methodologies greatly increases the possibilities to find a biocatalyst that can be suitable for a specific process.

DNA methyltransferase DNMT3A forms interaction networks with the CpG site and flanking sequence elements for efficient methylation.

Specific DNA methylation at CpG and non-CpG sites is essential for chromatin regulation. The DNA methyltransferase DNMT3A interacts with target sites surrounded by variable DNA sequences with its TRD and RD loops, but the functional necessity of these interactions is unclear. We investigated CpG and non-CpG methylation in a randomized sequence context using WT DNMT3A and several DNMT3A variants containing mutations at DNA-interacting residues. Our data revealed that the flanking sequence of target sites between the -2 and up to the +8 position modulates methylation rates >100-fold. Non-CpG methylation flanking preferences were even stronger and favor C(+1). R836 and N838 in concert mediate recognition of the CpG guanine. R836 changes its conformation in a flanking sequence-dependent manner and either contacts the CpG guanine or the +1/+2 flank, thereby coupling the interaction with both sequence elements. R836 suppresses activity at CNT sites but supports methylation of CAC substrates, the preferred target for non-CpG methylation of DNMT3A in cells. N838 helps to balance this effect and prevent the preference for C(+1) from becoming too strong. Surprisingly, we found L883 reduces DNMT3A activity despite being highly conserved in evolution. However, mutations at L883 disrupt the DNMT3A-specific DNA interactions of the RD loop, leading to altered flanking sequence preferences. Similar effects occur after the R882H mutation in cancer cells. Our data reveal that DNMT3A forms flexible and interdependent interaction networks with the CpG guanine and flanking residues that ensure recognition of the CpG and efficient methylation of the cytosine in contexts of variable flanking sequences.

Tuning Immobilized Commercial Lipase Preparations Features by Simple Treatment with Metallic Phosphate Salts.

Four commercial immobilized lipases biocatalysts have been submitted to modifications with different metal (zinc, cobalt or copper) phosphates to check the effects of this modification on enzyme features. The lipase preparations were Lipozyme®TL (TLL-IM) (lipase from Thermomyces lanuginose), Lipozyme®435 (L435) (lipase B from Candida antarctica), Lipozyme®RM (RML-IM), and LipuraSelect (LS-IM) (both from lipase from Rhizomucor miehei). The modifications greatly altered enzyme specificity, increasing the activity versus some substrates (e.g., TLL-IM modified with zinc phosphate in hydrolysis of triacetin) while decreasing the activity versus other substrates (the same preparation in activity versus R- or S- methyl mandelate). Enantiospecificity was also drastically altered after these modifications, e.g., LS-IM increased the activity versus the R isomer while decreasing the activity versus the S isomer when treated with copper phosphate. Regarding the enzyme stability, it was significantly improved using octyl-agarose-lipases. Using all these commercial biocatalysts, no significant positive effects were found; in fact, a decrease in enzyme stability was usually detected. The results point towards the possibility of a battery of biocatalysts, including many different metal phosphates and immobilization protocols, being a good opportunity to tune enzyme features, increasing the possibilities of having biocatalysts that may be suitable for a specific process.

Glycan processing in the Golgi as optimal information coding that constrains cisternal number and enzyme specificity.

Many proteins that undergo sequential enzymatic modification in the Golgi cisternae are displayed at the plasma membrane as cell identity markers. The modified proteins, called glycans, represent a molecular code. The fidelity of this glycan code is measured by how accurately the glycan synthesis machinery realizes the desired target glycan distribution for a particular cell type and niche. In this article, we construct a simplified chemical synthesis model to quantitatively analyse the trade-offs between the number of cisternae, and the number and specificity of enzymes, required to synthesize a prescribed target glycan distribution of a certain complexity to within a given fidelity. We find that to synthesize complex distributions, such as those observed in real cells, one needs to have multiple cisternae and precise enzyme partitioning in the Golgi. Additionally, for a fixed number of enzymes and cisternae, there is an optimal level of specificity (promiscuity) of enzymes that achieves the target distribution with high fidelity. The geometry of the fidelity landscape in the multidimensional space of the number and specificity of enzymes, inter-cisternal transfer rates, and number of cisternae provides a measure for robustness and identifies stiff and sloppy directions. Our results show how the complexity of the target glycan distribution and number of glycosylation enzymes places functional constraints on the Golgi cisternal number and enzyme specificity.

Preferential Self-interaction of DNA Methyltransferase DNMT3A Subunits Containing the R882H Cancer Mutation Leads to Dominant Changes of Flanking Sequence Preferences.

Somatic R882H DNMT3A mutations occur frequently in AML, but their pathogenic mechanism is unclear. As R882H mutations usually are heterozygous, wildtype (WT) and R882H subunits co-exist in affected cells. R882 is located in the RD interface of DNMT3A tetramers, which forms the DNA binding site. R882H causes strong changes in the flanking sequence preferences of DNMT3A. Here, we analyzed flanking sequence preferences for CGNNNN sites showing that most disfavored sites are methylated 4-5 fold slower by R882H than WT, while it methylates most preferred sites 2-fold faster. Overall, R882H was more active than WT at 13% and less active at 52% of all CGNNNN sites. We prepared mixed DNMT3A heterotetramers containing WT and R882H subunits and show that mixed complexes preferentially assemble with an R882H/R882H RD interface. Structural comparisons and MD simulations confirmed the conclusion that the R882H RD interface is more stable than that of WT, in part because H882 forms an inter-subunit contact in the RD interface, while R882 contacts the DNA. As the subunits at the RD interface contribute the two active centers to the DNMT3A tetramer, R882H characteristic flanking sequence preferences of DNMT3A were observed in mixed tetrameric complexes containing WT and R882H subunits, and they are not diluted by the "averaged" effects of mixed or WT interfaces. Hence, R882H has a dominant effect on the flanking sequence preferences and other catalytic properties of DNMT3A in samples containing WT and R882H subunits, which may explain its pathogenic effect in heterozygous state.

Structure, Activity and Function of the Dual Protein Lysine and Protein N-Terminal Methyltransferase METTL13.

METTL13 (also known as eEF1A-KNMT and FEAT) is a dual methyltransferase reported to target the N-terminus and Lys55 in the eukaryotic translation elongation factor 1 alpha (eEF1A). METTL13-mediated methylation of eEF1A has functional consequences related to translation dynamics and include altered rate of global protein synthesis and translation of specific codons. Aberrant regulation of METTL13 has been linked to several types of cancer but the precise mechanisms are not yet fully understood. In this article, the current literature related to the structure, activity, and function of METTL13 is systematically reviewed and put into context. The links between METTL13 and diseases, mainly different types of cancer, are also summarized. Finally, key challenges and opportunities for METTL13 research are pinpointed in a prospective outlook.

Mechanisms of inhibition of viral RNA replication by nucleotide analogs.

Nucleotide analogs are the cornerstone of direct acting antivirals used to control infection by RNA viruses. Here we review what is known about existing nucleotide/nucleoside analogs and the kinetics and mechanisms of RNA and DNA replication, with emphasis on the SARS-CoV-2 RNA dependent RNA polymerase (RdRp) in comparison to HIV reverse transcriptase and Hepatitis C RdRp. We demonstrate how accurate kinetic analysis reveals surprising results to explain the effectiveness of antiviral nucleoside analogs providing guidelines for the design of new inhibitors.

Loops around the Heme Pocket Have a Critical Role in the Function and Stability of BsDyP from Bacillus subtilis.

Bacillus subtilis BsDyP belongs to class I of the dye-decolorizing peroxidase (DyP) family of enzymes and is an interesting biocatalyst due to its high redox potential, broad substrate spectrum and thermostability. This work reports the optimization of BsDyP using directed evolution for improved oxidation of 2,6-dimethoxyphenol, a model lignin-derived phenolic. After three rounds of evolution, one variant was identified displaying 7-fold higher catalytic rates and higher production yields as compared to the wild-type enzyme. The analysis of X-ray structures of the wild type and the evolved variant showed that the heme pocket is delimited by three long conserved loop regions and a small α helix where, incidentally, the mutations were inserted in the course of evolution. One loop in the proximal side of the heme pocket becomes more flexible in the evolved variant and the size of the active site cavity is increased, as well as the width of its mouth, resulting in an enhanced exposure of the heme to solvent. These conformational changes have a positive functional role in facilitating electron transfer from the substrate to the enzyme. However, they concomitantly resulted in decreasing the enzyme's overall stability by 2 kcal mol-1, indicating a trade-off between functionality and stability. Furthermore, the evolved variant exhibited slightly reduced thermal stability compared to the wild type. The obtained data indicate that understanding the role of loops close to the heme pocket in the catalysis and stability of DyPs is critical for the development of new and more powerful biocatalysts: loops can be modulated for tuning important DyP properties such as activity, specificity and stability.

Structure, Activity and Function of the Suv39h1 and Suv39h2 Protein Lysine Methyltransferases.

SUV39H1 and SUV39H2 were the first protein lysine methyltransferases that were identified more than 20 years ago. Both enzymes introduce di- and trimethylation at histone H3 lysine 9 (H3K9) and have important roles in the maintenance of heterochromatin and gene repression. They consist of a catalytically active SET domain and a chromodomain, which binds H3K9me2/3 and has roles in enzyme targeting and regulation. The heterochromatic targeting of SUV39H enzymes is further enhanced by the interaction with HP1 proteins and repeat-associated RNA. SUV39H1 and SUV39H2 recognize an RKST motif with additional residues on both sides, mainly K4 in the case of SUV39H1 and G12 in the case of SUV39H2. Both SUV39H enzymes methylate different non-histone proteins including RAG2, DOT1L, SET8 and HupB in the case of SUV39H1 and LSD1 in the case of SUV39H2. Both enzymes are expressed in embryonic cells and have broad expression profiles in the adult body. SUV39H1 shows little tissue preference except thymus, while SUV39H2 is more highly expressed in the brain, testis and thymus. Both enzymes are connected to cancer, having oncogenic or tumor-suppressive roles depending on the tumor type. In addition, SUV39H2 has roles in the brain during early neurodevelopment.