Mechanism of RGD-conjugated nanodevice binding to its target protein integrin αVß3 by atomistic molecular dynamics and machine learning.

Active targeting strategies have been proposed to enhance the selective uptake of nanoparticles (NPs) by diseased cells, and recent experimental findings have proven the effectiveness of this approach. However, no mechanistic studies have yet revealed the atomistic details of the interactions between ligand-activated NPs and integrins. As a case study, here we investigate, by means of advanced molecular dynamics simulations (MD) and machine learning methods (namely equilibrium MD, binding free energy calculations and training of self-organized maps), the interaction of a cyclic-RGD-conjugated PEGylated TiO2 NP (the nanodevice) with the extracellular segment of integrin αVß3 (the target), the latter experimentally well-known to be over-expressed in several solid tumors. Firstly, we proved that the cyclic-RGD ligand binding to the integrin pocket is established and kept stable even in the presence of the cumbersome realistic model of the nanodevice. In this respect, the unsupervised machine learning analysis allowed a detailed comparison of the ligand/integrin binding in the presence and in the absence of the nanodevice, which unveiled differences in the chemical features. Then, we discovered that unbound cyclic RGDs conjugated to the NP largely contribute to the interactions between the nanodevice and the integrin. Finally, by increasing the density of cyclic RGDs on the PEGylated TiO2 NP, we observed a proportional enhancement of the nanodevice/target binding. All these findings can be exploited to achieve an improved targeting selectivity and cellular uptake, and thus a more successful clinical outcome.

Molecular Dynamics for the Optimal Design of Functionalized Nanodevices to Target Folate Receptors on Tumor Cells.

Atomistic details on the mechanism of targeting activity by biomedical nanodevices of specific receptors are still scarce in the literature, where mostly ligand/receptor pairs are modeled. Here, we use atomistic molecular dynamics (MD) simulations, free energy calculations, and machine learning approaches on the case study of spherical TiO2 nanoparticles (NPs) functionalized with folic acid (FA) as the targeting ligand of the folate receptor (FR). We consider different FA densities on the surface and different anchoring approaches, i.e., direct covalent bonding of FA γ-carboxylate or through polyethylene glycol spacers. By molecular docking, we first identify the lowest energy conformation of one FA inside the FR binding pocket from the X-ray crystal structure, which becomes the starting point of classical MD simulations in a realistic physiological environment. We estimate the binding free energy to be compared with the existing experimental data. Then, we increase complexity and go from the isolated FA to a nanosystem decorated with several FAs. Within the simulation time framework, we confirm the stability of the ligand-receptor interaction, even in the presence of the NP (with or without a spacer), and no significant modification of the protein secondary structure is observed. Our study highlights the crucial role played by the spacer, FA protonation state, and density, which are parameters that can be controlled during the nanodevice preparation step.

Assessing the Performance of Non-Equilibrium Thermodynamic Integration in Flavodoxin Redox Potential Estimation.

Flavodoxins are enzymes that contain the redox-active flavin mononucleotide (FMN) cofactor and play a crucial role in numerous biological processes, including energy conversion and electron transfer. Since the redox characteristics of flavodoxins are significantly impacted by the molecular environment of the FMN cofactor, the evaluation of the interplay between the redox properties of the flavin cofactor and its molecular surroundings in flavoproteins is a critical area of investigation for both fundamental research and technological advancements, as the electrochemical tuning of flavoproteins is necessary for optimal interaction with redox acceptor or donor molecules. In order to facilitate the rational design of biomolecular devices, it is imperative to have access to computational tools that can accurately predict the redox potential of both natural and artificial flavoproteins. In this study, we have investigated the feasibility of using non-equilibrium thermodynamic integration protocols to reliably predict the redox potential of flavodoxins. Using as a test set the wild-type flavodoxin from Clostridium Beijerinckii and eight experimentally characterized single-point mutants, we have computed their redox potential. Our results show that 75% (6 out of 8) of the calculated reaction free energies are within 1 kcal/mol of the experimental values, and none exceed an error of 2 kcal/mol, confirming that non-equilibrium thermodynamic integration is a trustworthy tool for the quantitative estimation of the redox potential of this biologically and technologically significant class of enzymes.

Dynamic Properties of the DNA Damage Response Mre11/Rad50 Complex.

DNA double-strand breaks (DSBs) are a significant threat to cell viability due to the induction of genome instability and the potential loss of genetic information. One of the key players for early DNA damage response is the conserved Mre11/Rad50 Nbs1/Xrs2 (MRN/X) complex, which is quickly recruited to the DNA's ruptured ends and is required for their tethering and their subsequent repair via different pathways. The MRN/X complex associates with several other proteins to exert its functions, but it also exploits sophisticated internal dynamic properties to orchestrate the several steps required to address the damage. In this review, we summarize the intrinsic molecular features of the MRN/X complex through biophysical, structural, and computational analyses in order to describe the conformational transitions that allow for this complex to accomplish its multiple functions.

Novel pathological variants of NHP2 affect N-terminal domain flexibility, protein stability, H/ACA Ribonucleoprotein (RNP) complex formation and telomerase activity.

Telomere biology disorders (TBDs) are characterized by short telomeres, premature aging, bone marrow failure and cancer predisposition. Germline mutations in NHP2, encoding for one component of the telomerase cofactor H/ACA RNA binding complex together with Dyskerin, NOP10 and GAR1, have been previously reported in rare cases of TBDs. Here, we report two novel NHP2 variants (NHP2-A39T and NHP2-T44M) identified in a compound heterozygous patient affected by premature aging, bone marrow failure/myelodysplastic syndrome and gastric cancer. Although still able to support cell viability, both variants reduce the levels of hTR, the telomerase RNA component, and telomerase activity, expanding the panel of NHP2 pathological variants. Furthermore, both variants fail to be incorporated in the H/ACA RNA binding complex when in competition with wild-type endogenous NHP2, and the lack of incorporation causes their drastic proteasomal degradation. By RoseTTAFold prediction followed by molecular dynamics simulations, we reveal a dramatic distortion of residues 33-41, which normally position on top of the NHP2 core, as the main defect of NHP2-A39T, and high flexibility and the misplacement of the N-terminal region (residues 1-24) in NHP2-T44M and, to a lower degree, in NHP2-A39T. Because deletion of amino acids 2-24 causes a reduction in NHP2 levels only in the presence of wild-type NHP2, while deletion of amino acids 2-38 completely disrupts NHP2 stability, we propose that the two variants are mis-incorporated into the H/ACA binding complex due to the altered dynamics of the first 23 amino acids and/or the distortion of the residues 25-41 loop.

Recent Theoretical Insights into the Oxidative Degradation of Biopolymers and Plastics by Metalloenzymes.

Molecular modeling techniques have become indispensable in many fields of molecular sciences in which the details related to mechanisms and reactivity need to be studied at an atomistic level. This review article provides a collection of computational modeling works on a topic of enormous interest and urgent relevance: the properties of metalloenzymes involved in the degradation and valorization of natural biopolymers and synthetic plastics on the basis of both circular biofuel production and bioremediation strategies. In particular, we will focus on lytic polysaccharide monooxygenase, laccases, and various heme peroxidases involved in the processing of polysaccharides, lignins, rubbers, and some synthetic polymers. Special attention will be dedicated to the interaction between these enzymes and their substrate studied at different levels of theory, starting from classical molecular docking and molecular dynamics techniques up to techniques based on quantum chemistry.

N-Acetylcysteine Antagonizes NGF Activation of TrkA through Disulfide Bridge Interaction, an Effect Which May Contribute to Its Analgesic Activity.

N-acetylcysteine (NAC), a mucolytic agent and an antidote to acetaminophen intoxication, has been studied in experimental conditions and trials exploring its analgesic activity based on its antioxidant and anti-inflammatory properties. The purpose of this study is to investigate additional mechanisms, namely, the inhibition of nerve growth factor (NGF) and the activation of the Tropomyosin receptor kinase A (TrkA) receptor, which is responsible for nociception. In silico studies were conducted to evaluate dithiothreitol and NAC's interaction with TrkA. We also measured the autophosphorylation of TrkA in SH-SY5Y cells via ELISA to assess NAC's in vitro activity against NGF-induced TrkA activation. The in silico and in vitro tests show that NAC interferes with NGF-induced TrkA activation. In particular, NAC breaks the disulfide-bound Cys 300-345 of TrkA, perturbing the NGF-TrkA interaction and producing a rearrangement of the binding site, inducing a consequent loss of their molecular recognition and spatial reorganization, which are necessary for the induction of the autophosphorylation process. The latter was inhibited by 40% using 20 mM NAC. These findings suggest that NAC could have a role as a TrkA antagonist, an action that may contribute to the activity and use of NAC in various pain states (acute, chronic, nociplastic) sustained by NGF hyperactivity and/or accompanied by spinal cord sensitization.

Molecular Dynamics Simulations Reveal Structural Interconnections within Sec14-PH Bipartite Domain from Human Neurofibromin.

Neurofibromin, the main RasGAP in the nervous system, is a 2818 aa protein with several poorly characterized functional domains. Mutations in the NF1-encoding gene lead to an autosomal dominant syndrome, neurofibromatosis, with an incidence of 1 out of 3000 newborns. Missense mutations spread in the Sec14-PH-encoding sequences as well. Structural data could not highlight the defect in mutant Sec14-PH functionality. By performing molecular dynamics simulations at different temperatures, we found that the lid-lock is fundamental for the structural interdependence of the NF1 bipartite Sec14-PH domain. In fact, increased flexibility in the lid-lock loop, observed for the K1750Δ mutant, leads to disconnection of the two subdomains and can affect the stability of the Sec14 subdomain.

AMPK Phosphorylation Is Controlled by Glucose Transport Rate in a PKA-Independent Manner.

To achieve growth, microbial organisms must cope with stresses and adapt to the environment, exploiting the available nutrients with the highest efficiency. In Saccharomyces cerevisiae, Ras/PKA and Snf1/AMPK pathways regulate cellular metabolism according to the supply of glucose, alternatively supporting fermentation or mitochondrial respiration. Many reports have highlighted crosstalk between these two pathways, even without providing a comprehensive mechanism of regulation. Here, we show that glucose-dependent inactivation of Snf1/AMPK is independent from the Ras/PKA pathway. Decoupling glucose uptake rate from glucose concentration, we highlight a strong coordination between glycolytic metabolism and Snf1/AMPK, with an inverse correlation between Snf1/AMPK phosphorylation state and glucose uptake rate, regardless of glucose concentration in the medium. Despite fructose-1,6-bisphosphate (F1,6BP) being proposed as a glycolytic flux sensor, we demonstrate that glucose-6-phosphate (G6P), and not F1,6BP, is involved in the control of Snf1/AMPK phosphorylation state. Altogether, this study supports a model by which Snf1/AMPK senses glucose flux independently from PKA activity, and thanks to conversion of glucose into G6P.

The Vault Nanoparticle: A Gigantic Ribonucleoprotein Assembly Involved in Diverse Physiological and Pathological Phenomena and an Ideal Nanovector for Drug Delivery and Therapy.

The vault nanoparticle is a eukaryotic ribonucleoprotein complex consisting of 78 individual 97 kDa-"major vault protein" (MVP) molecules that form two symmetrical, cup-shaped, hollow halves. It has a huge size (72.5 × 41 × 41 nm) and an internal cavity, wherein the vault poly(ADP-ribose) polymerase (vPARP), telomerase-associated protein-1 (TEP1), and some small untranslated RNAs are accommodated. Plenty of literature reports on the biological role(s) of this nanocomplex, as well as its involvement in diseases, mostly oncological ones. Nevertheless, much has still to be understood as to how vault participates in normal and pathological mechanisms. In this comprehensive review, current understanding of its biological roles is discussed. By different mechanisms, vault's individual components are involved in major cellular phenomena, which result in protection against cellular stresses, such as DNA-damaging agents, irradiation, hypoxia, hyperosmotic, and oxidative conditions. These diverse cellular functions are accomplished by different mechanisms, mainly gene expression reprogramming, activation of proliferative/prosurvival signaling pathways, export from the nucleus of DNA-damaging drugs, and import of specific proteins. The cellular functions of this nanocomplex may also result in the onset of pathological conditions, mainly (but not exclusively) tumor proliferation and multidrug resistance. The current understanding of its biological roles in physiological and pathological processes should also provide new hints to extend the scope of its exploitation as a nanocarrier for drug delivery.

Rational Design of Fe2 (µ-PR2 )2 (L)6 Coordination Compounds Featuring Tailored Potential Inversion.

It was recently discovered that some redox proteins can thermodynamically and spatially split two incoming electrons towards different pathways, resulting in the one-electron reduction of two different substrates, featuring reduction potential respectively higher and lower than the parent reductant. This energy conversion process, referred to as electron bifurcation, is relevant not only from a biochemical perspective, but also for the ground-breaking applications that electron-bifurcating molecular devices could have in the field of energy conversion. Natural electron-bifurcating systems contain a two-electron redox centre featuring potential inversion (PI), i. e. with second reduction easier than the first. With the aim of revealing key factors to tailor the span between first and second redox potentials, we performed a systematic density functional study of a 26-molecule set of models with the general formula Fe2 (µ-PR2 )2 (L)6 . It turned out that specific features such as i) a Fe-Fe antibonding character of the LUMO, ii) presence of electron-donor groups and iii) low steric congestion in the Fe's coordination sphere, are key ingredients for PI. In particular, the synergic effects of i)-iii) can lead to a span between first and second redox potentials larger than 700 mV. More generally, the "molecular recipes" herein described are expected to inspire the synthesis of Fe2 P2 systems with tailored PI, of primary relevance to the design of electron-bifurcating molecular devices.

Functional and structural insights into the MRX/MRN complex, a key player in recognition and repair of DNA double-strand breaks.

Chromosomal DNA double-strand breaks (DSBs) are potentially lethal DNA lesions that pose a significant threat to genome stability and therefore need to be repaired to preserve genome integrity. Eukaryotic cells possess two main mechanisms for repairing DSBs: non-homologous end-joining (NHEJ) and homologous recombination (HR). HR requires that the 5' terminated strands at both DNA ends are nucleolytically degraded by a concerted action of nucleases in a process termed DNA-end resection. This degradation leads to the formation of 3'-ended single-stranded DNA (ssDNA) ends that are essential to use homologous DNA sequences for repair. The evolutionarily conserved Mre11-Rad50-Xrs2/NBS1 complex (MRX/MRN) has enzymatic and structural activities to initiate DSB resection and to maintain the DSB ends tethered to each other for their repair. Furthermore, it is required to recruit and activate the protein kinase Tel1/ATM, which plays a key role in DSB signaling. All these functions depend on ATP-regulated DNA binding and nucleolytic activities of the complex. Several structures have been obtained in recent years for Mre11 and Rad50 subunits from archaea, and a few from the bacterial and eukaryotic orthologs. Nevertheless, the mechanism of activation of this protein complex is yet to be fully elucidated. In this review, we focused on recent biophysical and structural insights on the MRX complex and their interplay.

DNA binding modes influence Rap1 activity in the regulation of telomere length and MRX functions at DNA ends.

The cellular response to DNA double-strand breaks (DSBs) is initiated by the Mre11-Rad50-Xrs2 (MRX) complex that has structural and catalytic functions. MRX association at DSBs is counteracted by Rif2, which is known to interact with Rap1 that binds telomeric DNA through two tandem Myb-like domains. Whether and how Rap1 acts at DSBs is unknown. Here we show that Rif2 inhibits MRX association to DSBs in a manner dependent on Rap1, which binds to DSBs and promotes Rif2 association to them. Rap1 in turn can negatively regulate MRX function at DNA ends also independently of Rif2. In fact, a characterization of Rap1 mutant variants shows that Rap1 binding to DNA through both Myb-like domains results in formation of Rap1-DNA complexes that control MRX functions at both DSBs and telomeres primarily through Rif2. By contrast, Rap1 binding to DNA through a single Myb-like domain results in formation of high stoichiometry complexes that act at DNA ends mostly in a Rif2-independent manner. Altogether these findings indicate that the DNA binding modes of Rap1 influence its functional properties, thus highlighting the structural plasticity of this protein.

The ATP-bound conformation of the Mre11-Rad50 complex is essential for Tel1/ATM activation.

Activation of the checkpoint protein Tel1 requires the Mre11-Rad50-Xrs2 (MRX) complex, which recruits Tel1 at DNA double-strand breaks (DSBs) through direct interaction between Tel1 and Xrs2. However, in vitro Tel1 activation by MRX requires ATP binding to Rad50, suggesting a role also for the MR subcomplex in Tel1 activation. Here we describe two separation-of-functions alleles, mre11-S499P and rad50-A78T, which we show to specifically affect Tel1 activation without impairing MRX functions in DSB repair. Both Mre11-S499P and Rad50-A78T reduce Tel1-MRX interaction leading to poor Tel1 association at DSBs and consequent loss of Tel1 activation. The Mre11-S499P variant reduces Mre11-Rad50 interaction, suggesting an important role for MR complex formation in Tel1 activation. Molecular dynamics simulations show that the wild type MR subcomplex bound to ATP lingers in a tightly 'closed' conformation, while ADP presence leads to the destabilization of Rad50 dimer and of Mre11-Rad50 association, both events being required for MR conformational transition to an open state. By contrast, MRA78T undertakes complex opening even if Rad50 is bound to ATP, indicating that defective Tel1 activation caused by MRA78T results from destabilization of the ATP-bound conformational state.

Local unwinding of double-strand DNA ends by the MRX complex promotes Exo1 processing activity.

Homologous recombination is initiated by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs), which involves different nucleases including the Mre11-Rad50-Xrs2 (MRX) complex and the Exonuclease 1 (Exo1). The characterization of a novel mutation in Mre11 causing accelerated DSB resection has allowed to show that MRX facilitates DNA end processing by Exo1 through local unwinding of double-stranded DNA ends.

The MRX complex regulates Exo1 resection activity by altering DNA end structure.

Homologous recombination is triggered by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs). DSB resection requires the Mre11-Rad50-Xrs2 (MRX) complex, which promotes the activity of Exo1 nuclease through a poorly understood mechanism. Here, we describe the Mre11-R10T mutant variant that accelerates DSB resection compared to wild-type Mre11 by potentiating Exo1-mediated processing. This increased Exo1 resection activity leads to a decreased association of the Ku complex to DSBs and an enhanced DSB resection in G1, indicating that Exo1 has a direct function in preventing Ku association with DSBs. Molecular dynamics simulations show that rotation of the Mre11 capping domains is able to induce unwinding of double-strand DNA (dsDNA). The R10T substitution causes altered orientation of the Mre11 capping domain that leads to persistent melting of the dsDNA end. We propose that MRX creates a specific DNA end structure that promotes Exo1 resection activity by facilitating the persistence of this nuclease on the DSB ends, uncovering a novel MRX function in DSB resection.

Evolutionary Analysis Provides Insight Into the Origin and Adaptation of HCV.

Hepatitis C virus (HCV) belongs to the Hepacivirus genus and is genetically heterogeneous, with seven major genotypes further divided into several recognized subtypes. HCV origin was previously dated in a range between ∼200 and 1000 years ago. Hepaciviruses have been identified in several domestic and wild mammals, the largest viral diversity being observed in bats and rodents. The closest relatives of HCV were found in horses/donkeys (equine hepaciviruses, EHV). However, the origin of HCV as a human pathogen is still an unsolved puzzle. Using a selection-informed evolutionary model, we show that the common ancestor of extant HCV genotypes existed at least 3000 years ago (CI: 3192-5221 years ago), with the oldest genotypes being endemic to Asia. EHV originated around 1100 CE (CI: 291-1640 CE). These time estimates exclude that EHV transmission was mainly sustained by widespread veterinary practices and suggest that HCV originated from a single zoonotic event with subsequent diversification in human populations. We also describe a number of biologically important sites in the major HCV genotypes that have been positively selected and indicate that drug resistance-associated variants are significantly enriched at positively selected sites. HCV exploits several cell-surface molecules for cell entry, but only two of these (CD81 and OCLN) determine the species-specificity of infection. Herein evolutionary analyses do not support a long-standing association between primates and hepaciviruses, and signals of positive selection at CD81 were only observed in Chiroptera. No evidence of selection was detected for OCLN in any mammalian order. These results shed light on the origin of HCV and provide a catalog of candidate genetic modulators of HCV phenotypic diversity.

Structurally distinct Mre11 domains mediate MRX functions in resection, end-tethering and DNA damage resistance.

Sae2 cooperates with the Mre11-Rad50-Xrs2 (MRX) complex to initiate resection of DNA double-strand breaks (DSBs) and to maintain the DSB ends in close proximity to allow their repair. How these diverse MRX-Sae2 functions contribute to DNA damage resistance is not known. Here, we describe mre11 alleles that suppress the hypersensitivity of sae2Δ cells to genotoxic agents. By assessing the impact of these mutations at the cellular and structural levels, we found that all the mre11 alleles that restore sae2Δ resistance to both camptothecin and phleomycin affect the Mre11 N-terminus and suppress the resection defect of sae2Δ cells by lowering MRX and Tel1 association to DSBs. As a consequence, the diminished Tel1 persistence potentiates Sgs1-Dna2 resection activity by decreasing Rad9 association to DSBs. By contrast, the mre11 mutations restoring sae2Δ resistance only to phleomycin are located in Mre11 C-terminus and bypass Sae2 function in end-tethering but not in DSB resection, possibly by destabilizing the Mre11-Rad50 open conformation. These findings unmask the existence of structurally distinct Mre11 domains that support resistance to genotoxic agents by mediating different processes.

Structural characterization of the nitrogenase molybdenum-iron protein with the substrate acetylene trapped near the active site.

The biological reduction of dinitrogen (N2) to ammonia is catalyzed by the complex metalloenzyme nitrogenase. Structures of the nitrogenase component proteins, Iron (Fe) protein and Molybdenum­iron (MoFe) protein, and the stabilized complexes these component proteins, have been determined, providing a foundation for a number of fundamental aspects of the complicated catalytic mechanism. The reduction of dinitrogen to ammonia is a complex process that involves the binding of N2 followed by reduction with multiple electrons and protons. Electron transfer into nitrogenase is typically constrained to the unique electron donor, the Fe protein. These constraints have prevented structural characterization of the active site with bound substrate. Recently it has been realized that selected amino acid substitutions in the environment of the active site metal cluster (Iron­molybdenum cofactor, FeMo-co) allow substrates to persist even in the resting state. Reported here is a 1.70Å crystal structure of a nitrogenase MoFe protein α-96Arg➔Gln variant with the alternative substrate acetylene trapped in a channel in close proximity to FeMo-co. Complementary theoretical calculations support the validity of the acetylene interaction at this site and is also consistent with more favorable interactions in the variant MoFe protein compared to the native MoFe protein. This work represents the first structural evidence of a substrate trapped in the nitrogenase MoFe protein and is consistent with earlier assignments of proposed substrate pathways and substrate binding sites deduced from biochemical, spectroscopic, and theoretical studies.

Epigallocatechin-3-gallate and related phenol compounds redirect the amyloidogenic aggregation pathway of ataxin-3 towards non-toxic aggregates and prevent toxicity in neural cells and Caenorhabditis elegans animal model.

The protein ataxin-3 (ATX3) triggers an amyloid-related neurodegenerative disease when its polyglutamine stretch is expanded beyond a critical threshold. We formerly demonstrated that the polyphenol epigallocatechin-3-gallate (EGCG) could redirect amyloid aggregation of a full-length, expanded ATX3 (ATX3-Q55) towards non-toxic, soluble, SDS-resistant aggregates. Here, we have characterized other related phenol compounds, although smaller in size, i.e. (-)-epigallocatechin gallate (EGC), and gallic acid (GA). We analysed the aggregation pattern of ATX3-Q55 and of the N-terminal globular Josephin domain (JD) by assessing the time course of the soluble protein, as well its structural features by FTIR and AFM, in the presence and the absence of the mentioned compounds. All of them redirected the aggregation pattern towards soluble, SDS-resistant aggregates. They also prevented the appearance of ordered side-chain hydrogen bonding in ATX3-Q55, which is the hallmark of polyQ-related amyloids. Molecular docking analyses on the JD highlighted three interacting regions, including the central, aggregation-prone one. All three compounds bound to each of them, although with different patterns. This might account for their capability to prevent amyloidogenesis. Saturation transfer difference NMR experiments also confirmed EGCG and EGC binding to monomeric JD. ATX3-Q55 pre-incubation with any of the three compounds prevented its calcium-influx-mediated cytotoxicity towards neural cells. Finally, all the phenols significantly reduced toxicity in a transgenic Caenorhabditis elegans strain expressing an expanded ATX3. Overall, our results show that the three polyphenols act in a substantially similar manner. GA, however, might be more suitable for antiamyloid treatments due to its simpler structure and higher chemical stability.