Injectable Liposome-Loaded Hydrogel Formulations with Controlled Release of Curcumin and α-Tocopherol for Dental Tissue Engineering.

An injectable hydrogel formulation is developed utilizing low- and high-molecular-weight chitosan (LCH and HCH) incorporated with curcumin and α-tocopherol-loaded liposomes (Lip/Cur+Toc). Cur and Toc releases are delayed within the hydrogels. The injectability of hydrogels is proved via rheological analyses. In vitro studies are conducted using human dental pulp stem cells (hDPSCs) and human gingival fibroblasts (hGFs) to examine the biological performance of the hydrogels toward endodontics and periodontics, respectively. The viability of hDPSCs treated with the hydrogels with Lip/Cur+Toc is the highest till day 14, compared to the neat hydrogels. During odontogenic differentiation tests, alkaline phosphatase (ALP) enzyme activity of hDPSCs is induced in the Cur-containing groups. Biomineralization is enhanced mostly with Lip/Cur+Toc incorporation. The viability of hGFs is the highest in HCH combined with Lip/Cur+Toc while wound healing occurs almost 100% in both (Lip/Cur+Toc@LCH and Lip/Cur+Toc@HCH) after 2 days. Antioxidant activity of Lip/Cur+Toc@LCH on hGFs is significantly the highest among the groups. Antimicrobial tests demonstrate that Lip/Cur+Toc@LCH is more effective against Escherichia coli whereas so is Lip/Cur+Toc@HCH against Staphylococcus aureus. The antimicrobial mechanism of the hydrogels is investigated for the first time through various computational models. LCH and HCH loaded with Lip/Cur+Toc are promising candidates with multi-functional features for endodontics and periodontics.

Efficient Calculation of the Dispersion Energy for Multireference Systems with Cholesky Decomposition: Application to Excited-State Interactions.

Accurate and efficient prediction of dispersion interactions in excited-state complexes poses a challenge due to the complex nature of electron correlation effects that need to be simultaneously considered. We propose an algorithm for computing the dispersion energy in nondegenerate ground- or excited-state complexes with arbitrary spin. The algorithm scales with the fifth power of the system size due to employing Cholesky decomposition of Coulomb integrals and a recently developed recursive formula for density response functions of the monomers. As a numerical illustration, we apply the new algorithm in the framework of multiconfigurational symmetry adapted perturbation theory, SAPT(MC), to study interactions in dimers with localized excitons. The SAPT(MC) analysis reveals that the dispersion energy may be the main force stabilizing excited-state dimers.

Insights into generalization of the rate-limiting steps of the dehalogenation by LinB and DhaA: A computational approach.

LinB and DhaA are well-known haloalkane dehalogenases (HLDs) capable of converting a plethora of halogenated alkanes, also those considered persistent pollutants. The dehalogenation reaction that these two enzymes catalyze has been studied to determine its rate-limiting step (rls) for the last two decades now. As a result, it has been determined that HLDs can show different rate-limiting steps for individual substrates, and at this point we do not have a basis for any generalization in this matter. Therefore, in this work we aimed at gaining insights into the enzymatic dehalogenation of selected dibromo- and bromochloro-ethanes and propanes by LinB and DhaA using computational approach to determine whether defined structural similarities of the substrates result in a unified mechanism and the same rls. By predicting halogen binding isotope effects (BIEs) as well as computing interaction energy for each HLD-ligand complex the nature of the protein-ligand interactions has been characterized. Furthermore, C and Br kinetic isotope effects (KIEs) as well as the minimum free energy paths (MFEPs) were computed to investigate the chemical reaction for the selected systems. Accuracy of the approach and robustness of the computational predictions were validated by measuring KIEs on the selected reactions. Overall results strongly indicate that any generalization with respect to the enzymatic process involving various ligands in the case of DhaA is impossible, even if the considered ligands are structurally very similar as those analyzed in the present study. Moreover, even small structural differences such as changing of one of the (non-leaving) halogen substituents may lead to significant changes in the enzymatic process and result in a different rls in the case of LinB. It has also been demonstrated that KIEs themselves cannot be used as rls indicators in the reactions catalyzed by the studied HLDs.

Influence of Association on Binding of Disaccharides to YKL-39 and hHyal-1 Enzymes.

Disaccharide complexes have been shown experimentally to be useful for drug delivery or as an antifouling surface biofilm, and are promising drug-encapsulation and delivery candidates. Although such complexes are intended for medical applications, to date no studies at the molecular level have been devoted to the influence of complexation on the enzymatic decomposition of polysaccharides. A theoretical approach to this problem has been hampered by the lack of a suitable computational tool for binding such non-covalent complexes to enzymes. Herein, we combine quantum-mechanical calculations of disaccharides complexes with a nonstandard docking GaudiMM engine that can perform such a task. Our results on four different complexes show that they are mostly stabilized by electrostatic interactions and hydrogen bonds. This strong non-covalent stabilization demonstrates the studied complexes are some excellent candidates for self-assembly smart materials, useful for drug encapsulation and delivery. Their advantage lies also in their biocompatible and biodegradable character.

Dispersion Interactions in Exciton-Localized States. Theory and Applications to π-π* and n-π* Excited States.

We address the problem of intermolecular interaction energy calculations in molecular complexes with localized excitons. Our focus is on the correct representation of the dispersion energy. We derive an extended Casimir-Polder formula for direct computation of this contribution through second order in the intermolecular interaction operator V̂. An alternative formula, accurate to infinite order in V̂, is derived within the framework of the adiabatic connection (AC) theory. We also propose a new parametrization of the VV10 nonlocal correlation density functional, so that it corrects the CASSCF energy for the dispersion contribution and can be applied to excited-state complexes. A numerical investigation is carried out for benzene, pyridine, and peptide complexes with the local exciton corresponding to the lowest π-π* or n- π* states. The extended Casimir-Polder formula is implemented in the framework of multiconfigurational symmetry-adapted perturbation theory, SAPT(MC). A SAPT(MC) analysis shows that the creation of a localized exciton affects mostly the electrostatic component of the interaction energy of investigated complexes. Nevertheless, the changes in Pauli repulsion and dispersion energies cannot be neglected. We verify the performance of several perturbation- and AC-based methods. Best results are obtained with a range-separated variant of an approximate AC approach employing extended random phase approximation and CASSCF wave functions.

Theoretical Studies of Cyanophycin Dipeptides as Inhibitors of Tyrosinases.

The three-dimensional structure of tyrosinase has been crystallized from many species but not from Homo sapiens. Tyrosinase is a key enzyme in melanin biosynthesis, being an important target for melanoma and skin-whitening cosmetics. Several studies employed the structure of tyrosinase from Agaricus bisporus as a model enzyme. Recently, 98% of human genome proteins were elucidated by AlphaFold. Herein, the AlphaFold structure of human tyrosinase and the previous model were compared. Moreover, tyrosinase-related proteins 1 and 2 were included, along with inhibition studies employing kojic and cinnamic acids. Peptides are widely studied for their inhibitory activity of skin-related enzymes. Cyanophycin is an amino acid polymer produced by cyanobacteria and is built of aspartic acid and arginine; arginine can be also replaced by other amino acids. A new set of cyanophycin-derived dipeptides was evaluated as potential inhibitors. Aspartate-glutamate showed the strongest interaction and was chosen as a leading compound for future studies.

Dispersion Interactions between Molecules in and out of Equilibrium Geometry: Visualization and Analysis.

The London dispersion interactions between systems undergoing bond breaking, twisting, or compression are not well studied due to the scarcity and the high computational cost of methods being able to describe both the dynamic correlation and the multireference character of the system. Recently developed methods based on the Generalized Valence Bond wave function, such as EERPA-GVB and SAPT(GVB) (SAPT = symmetry-adapted perturbation theory), allow one to accurately compute and analyze noncovalent interactions between multireference systems. Here, we augment this analysis by introducing a local indicator for dispersion interactions inspired by Mata and Wuttke's Dispersion Interaction Density [ J. Comput. Chem. 2017, 38, 15-23] applied on top of an EERPA-GVB computation. Using a few model systems, we show what insights into the nature and evolution of the dispersion interaction during bond breaking and twisting such an approach is able to offer. The new indicator can be used at a minimal cost additional to an EERPA-GVB computation and can be complemented by an energy decomposition employing the SAPT(GVB) method. We explain the physics behind the initial increase, followed by a decrease in the interaction of linear molecules upon bond stretching. Namely, the elongation of covalent bonds leads to the enhancement of attractive dispersion interactions. For even larger bond lengths, this effect is canceled by the increase of the repulsive exchange forces resulting in a suppression of the interaction and finally leading to repulsion between monomers.

How Much Dispersion Energy Is Included in the Multiconfigurational Interaction Energy?

We demonstrate how to quantify the amount of dispersion interaction recovered by supermolecular calculations with the multiconfigurational self-consistent field (MCSCF) wave functions. For this purpose, we present a rigorous derivation which connects the portion of dispersion interaction captured by the assumed wave function model-the residual dispersion interaction-with the size of the active space. Based on the obtained expression for the residual dispersion contribution, we propose a dispersion correction for the MCSCF that avoids correlation double counting. Numerical demonstration for model four-electron dimers in both ground and excited states described with the complete active space self-consistent field (CASSCF) reference serves as a proof-of-concept for the method. Accurate results, largely independent of the size of the active space, are obtained. For many-electron systems, routine CASSCF interaction energy calculations recover a tiny fraction of the full second-order dispersion energy. We found that the residual dispersion is non-negligible only for purely dispersion-bound complexes.

Evolved Fusarium oxysporum laccase expressed in Saccharomyces cerevisiae.

Fusarium oxysporum laccase was functionally expressed in Saccharomyces cerevisiae and engineered towards higher expression levels and higher reactivity towards 2,6-dimethoxyphenol, that could be used as a mediator for lignin modification. A combination of classical culture optimization and protein engineering led to around 30 times higher activity in the culture supernatant. The winner mutant exhibited three times lower Km, four times higher kcat and ten times higher catalytic efficiency than the parental enzyme. The strategy for laccase engineering was composed of a combination of random methods with a rational approach based on QM/MM MD studies of the enzyme complex with 2,6-dimethoxyphenol. Laccase mediator system with 2,6-dimethoxyphenol caused fulvic acids release from biosolubilized coal.

Long-range-corrected multiconfiguration density functional with the on-top pair density.

We propose a multiconfiguration density functional combining a short-range density functional approximation with a novel long-range correction for dynamic correlation effects. The correction is derived from the adiabatic connection formalism so that the resulting functional requires access only to one- and two-electron reduced density matrices of the system. In practice, the functional is formulated for wavefunctions of the complete active space (CAS) type and the short-range density functional part is made dependent on the on-top pair density via auxiliary spin densities. The latter allows for reducing the self-interaction and the static correlation errors without breaking the spin symmetry. We study the properties and the performance of the non-self-consistent variant of the method, termed lrAC0-postCAS. Numerical demonstration on a set of dissociation energy curves and excitation energies shows that lrAC0-postCAS provides accuracy comparable with more computationally expensive ab initio rivals.

Effect of modified atmosphere packaging on quality of bread with amaranth flour addition.

The study of the effect of the modified atmosphere packaging on quality of the bread was done after replacing wheat flour with amaranth flour in replacement of wheat flour for 0, 5, 10 and 15% by weight. The bread was stored for one, three and seven days in the ATM, 100% N2 and 30% CO2. The study proved the 30% CO2 modified atmosphere to be the optimal storage condition. It allowed to preserve volume, water content and contributed at least to increase in hardness of bread, which with the addition of amaranth flour decreased. The colour of bread during storage was characterised by the increase in L* parameter and decrease in a* and b*. For the decreased storage time, the effect was opposite. The replacement by 5% of amaranth flour increased the polyphenol content and did not affect bread quality.

On the origins of stereoselectivity in the aminocatalytic remote alkylation of 5-alkylfurfurals.

In the manuscript, computational studies on the remote alkylation of 5-alkylfurfurals proceeding via formation of the corresponding trienamine intermediate are presented. By the means of density functional theory (DFT) calculations and the symmetry-adapted perturbation theory (SAPT) method, interesting insights into the mechanism of the reaction have been provided explaining the influence and contribution of different molecular interactions on the observed reactivity as well as on the enantio- and diastereoselectivity of the process. The studies have been extended to the thiophene analogue of the starting furfural derivative and the results obtained verified experimentally.

Molecular Insights into the Substrate-Assisted Mechanism of Viral DNA 3'-End Processing in Intasome of Prototype Foamy Virus Integrase from Molecular Dynamic and QM/MM Studies.

Integrases participate in two important steps for virus replication such as 3'-end processing of viral DNA ( vDNA) and nuclear entry of host DNA ( hDNA). In this work, insight into the structural changes in intasome of prototype foamy virus integrase (PFV-IN) complexed with vDNA from classical molecular dynamic (MD) simulations are done. Analysis of the results reveal the existence of alternative conformations of the enzyme active site indicating that the 3'-end processing reaction can occur according to three different pathways and taking place with the possible participation of aspartate 185 of a neighboring phosphate group or involving an internal phosphate group of the substrate. In this work, one of them, the so-called substrate-assisted mechanism was explored by QM/MM methods. The free energy barriers of 34.4 kcal mol-1 for the first and 35.3 kcal mol-1 for the second step of reaction computed with free energy perturbation (FEP) methods at the M06-2X/AMBER level show that 3'-end processing has to proceed via a different mechanism than studied herein. Nevertheless, the obtained results are in good agreement with the experimental observations that the substitution of the key atom for this mechanism, oxygen by sulfur, did not influence the catalysis. Additionally, the obtained mechanism reveals significant similarities to the previously studied substrate-assisted mechanism in twister ribozyme. The possible role of Mg2+ in the active site is discussed.

2-OMe-lysophosphatidylcholine analogues are GPR119 ligands and activate insulin secretion from ßTC-3 pancreatic cells: Evaluation of structure-dependent biological activity.

GPR119 receptor has been proposed as a metabolic regulator playing a pivotal role in the modulation of glucose homeostasis in type 2 diabetes. GPR119 was identified on pancreatic ß cells and its ligands have the ability to enhance glucose-stimulated insulin secretion (GSIS). Lysophosphatidylcholine (LPC) was shown to potentiate GSIS and our present studies indicate that 2-methoxy-lysophosphatidylcholine (2-OMe-LPC) analogues, unable to undergo 1→2 acyl migration, stimulate GSIS from murine ßTC-3 pancreatic cells even more efficiently. Moreover, biological assays in engineered Tango™ GPR119-bla U2OS cells were carried out to ascertain the agonist activity of 2-OMe-LPC at GPR119. 2-OMe-LPC possessing in sn-1 position the residues of myristic, palmitic, stearic and oleic acid were also evaluated as factors regulating [Ca2+]i mobilization and cAMP levels. Extension of these studies to R- and S-enantiomers of 14:0 2-OMe-LPC revealed that the overall impact on GSIS does not depend on chirality, however, the intracellular calcium mobilization data show that the R enantiomer is significantly more active than S one. Taking into account differences in chemical structure between various native LPCs and their 2-methoxy counterparts the possible binding mode of 2-OMe-LPC to the GPR119 receptor was determined using molecular modeling approach.

Oxygen binding isotope effects of triazole-based HIV-1 reverse transcriptase inhibitors indicate the actual binding site.

Binding isotope effects (BIEs) associated with binding of four triazole-based ligands to HIV-1 reverse transcriptase have been calculated at the QM/MM MD level of theory. Two main binding sites: allosteric cavity and RNase H active site, as well as three other sites reported in the literature (the Knuckles, the NNRTI Adjacent, and Incoming Nucleotide Binding) have been considered. The interactions between inhibitors and these protein sites have been quantified by binding free energies obtained from free energy perturbation (FEP) calculations, supported by interaction energy analysis. It has been shown that binding in the allosteric cavity can be distinguished from binding to other sites based on BIEs as it is associated with normal 18O-BIEs of the carbonyl oxygen atom while binding to RNase H active site is characterized by inverse binding isotope effect (18O-BIE < 1). For other sites 18O-BIEs close to unity are predicted. This information points to oxygen binding isotope effects of carbonyl group as indicative of the actual binding site of studied inhibitors.

Insights into the role of methionine synthase in the universal 13C depletion in O- and N-methyl groups of natural products.

Many O-methyl and N-methyl groups in natural products are depleted in 13C relative to the rest of the molecule. These methyl groups are derived from the C-1 tetrahydrofolate pool via l-methionine, the principle donor of methyl units. Depletion could occur at a number of steps in the pathway. We have tested the hypothesis that methionine biosynthesis is implicated in this depletion by using a combined experimental and theoretical approach. By using isotope ratio monitoring 13C NMR spectrometry to measure the position-specific distribution of 13C within l-methionine of natural origin, it is shown that the S-methyl group is depleted in 13C by ∼20‰ relative to the other positions in the molecule. In parallel, we have conducted a basic theoretical analysis of the reaction pathway of methionine synthase to assess whether the enzyme cobalamin-independent l-methionine synthase (EC 2.1.1.14)-that catalyzes the synthesis of l-methionine from 5-methyl-tetrahydrofolate and homocysteine-plays a role in causing this depletion. Calculation predicts a strong normal 13C kinetic isotope effect (1.087) associated with this enzyme. Hence, depletion in 13C in the S-methyl of l-methionine during biosynthesis can be identified as an important factor contributing to the general depletion seen in many O-methyl and N-methyl groups of natural products.

Lysophosphatidylcholine elicits intracellular calcium signaling in a GPR55-dependent manner.

The GPR55 signaling is fertile ground for drug discovery, however despite considerable research progress during the past 10 years, many open questions remain. The GPR55 pharmacology remains controversial, as many ligands have been reported with inconsistent results. Here, we show that various molecular species of lysophosphatidylcholine (LPC) elicit intracellular Ca2+ mobilization in GPR55-expressing PC-3 human prostate carcinoma cells. The response was even stronger than [Ca2+]i flux evoked by endogenous (OEA) and synthetic (Abn-CBD) agonists. Treatment with GPR55 antagonists CID16020046 and ML193 as well as the lipid raft disrupter methyl-ß-cyclodextrin strongly blunted LPC-induced calcium signal. Additionally, molecular modeling analysis revealed that LPC 16:0 and LPC 18:1 interact stronger with the receptor than to OEA. Identified electrostatic interactions between GPR55 residues and the ligands overlap with the binding site identified previously for lysophosphatidylinositol. Therefore, we prove that LPC is another GPR55-sensitive ligand. This finding is relevant in understanding lysophospolipids-mediated signaling and opens new avenues to develop therapeutic approach based on GPR55 targeting.

Dynamic and Electrostatic Effects on the Reaction Catalyzed by HIV-1 Protease.

HIV-1 Protease (HIV-1 PR) is one of the three enzymes essential for the replication process of HIV-1 virus, which explains why it has been the main target for design of drugs against acquired immunodeficiency syndrome (AIDS). This work is focused on exploring the proteolysis reaction catalyzed by HIV-1 PR, with special attention to the dynamic and electrostatic effects governing its catalytic power. Free energy surfaces for all possible mechanisms have been computed in terms of potentials of mean force (PMFs) within hybrid QM/MM potentials, with the QM subset of atoms described at semiempirical (AM1) and DFT (M06-2X) level. The results suggest that the most favorable reaction mechanism involves formation of a gem-diol intermediate, whose decomposition into the product complex would correspond to the rate-limiting step. The agreement between the activation free energy of this step with experimental data, as well as kinetic isotope effects (KIEs), supports this prediction. The role of the protein dynamic was studied by protein isotope labeling in the framework of the Variational Transition State Theory. The predicted enzyme KIEs, also very close to the values measured experimentally, reveal a measurable but small dynamic effect. Our calculations show how the contribution of dynamic effects to the effective activation free energy appears to be below 1 kcal·mol-1. On the contrary, the electric field created by the protein in the active site of the enzyme emerges as being critical for the electronic reorganization required during the reaction. These electrostatic properties of the active site could be used as a mold for future drug design.

DFT Studies of SN2 Dechlorination of Polychlorinated Biphenyls.

Nucleophilic dechlorination of all 209 PCBs congeners by ethylene glycol anion has been studied theoretically at the DFT level. The obtained Gibbs free energies of activation are in the range 7-22 kcal/mol. The reaction Gibbs free energies indicate that all reactions are virtually irreversible. Due to geometric constrains these reactions undergo rather untypical attack with attacking oxygen atom being nearly perpendicular to the attacked C-Cl bond. The most prone to substitution are chlorine atoms that occupy ortho- (2, 2', 6, 6') positions. These results provide extensive information on the PEG/KOH dependent PCBs degradation. They can also be used in further developments of reaction class transition state theory (RC-TST) for description of complex reactive systems encountered for example in combustion processes.

The use of plasmapheresis in a 4-year-old boy with toxic epidermal necrosis.

BACKGROUND: Toxic epidermal necrosis (TEN) is a severe and life-threatening syndrome manifesting as extensive necrosis of the epidermis and mucous membranes accompanied by systemic symptoms. The causative factor is exposure to drugs, such as sulphonamides, antiepileptic preparations, non-steroidal anti-inflammatory drugs and paracetamol. The mechanism leading to the development of lesions is unknown and there is no uniform strategy of management. CASE REPORT: A 4-year-old boy was admitted with late-stage TEN; he was additionally affected by partial atrophy of the corpus callosum, mental retardation and drug-resistant epilepsy. Three weeks before the first symptoms developed, antiepileptic treatment was widened with lamotrigine, which seems to be the causative factor of TEN. Since general and topical pharmacological therapy failed, plasmapheresis was applied and already after 2 cycles, the progression of lesions was inhibited and circulation was gradually stabilized. CONCLUSIONS: The use of plasmapheresis in a child with TEN can result in substantial improvement of the general and local condition. The case presented clearly indicates that standards of management of children with suspected or developed TEN should be urgently instituted.