Methods and Applications of In Silico Aptamer Design and Modeling

Aptamers are nucleic acid analogues of antibodies with high affinity to different targets, such as cells, viruses, proteins, inorganic materials, and coenzymes. Empirical approaches allow the design of in vitro aptamers that bind particularly to a target molecule with high affinity and selectivity. Theoretical methods allow significant expansion of the possibilities of aptamer design. In this study, we review theoretical and joint theoretical-experimental studies dedicated to aptamer design and modeling. We consider aptamers with different targets, such as proteins, antibiotics, organophosphates, nucleobases, amino acids, and drugs. During nucleic acid modeling and in silico design, a full set of in silico methods can be applied, such as docking, molecular dynamics (MD), and statistical analysis. The typical modeling workflow starts with structure prediction. Then, docking of target and aptamer is performed. Next, MD simulations are performed, which allows for an evaluation of the stability of aptamer/ligand complexes and determination of the binding energies with higher accuracy. Then, aptamer/ligand interactions are analyzed, and mutations of studied aptamers made. Subsequently, the whole procedure of molecular modeling can be reiterated. Thus, the interactions between aptamers and their ligands are complex and difficult to understand using only experimental approaches. Docking and MD are irreplaceable when aptamers are studied in silico.     

Modulation of Aβ42 Aggregation Kinetics and Pathway by Low-Molecular-Weight Inhibitors

The aggregation of amyloid-β 42 (Aβ42) is directly related to the pathogenesis of Alzheimer's disease. Here, we have investigated the early stages of the aggregation process, during which most of the cytotoxic species are formed. Aβ42 aggregation kinetics, characterized by the quantification of Aβ42 monomer consumption, were tracked by real-time solution NMR spectroscopy (RT-NMR) allowing the impact that low-molecular-weight (LMW) inhibitors and modulators exert on the aggregation process to be analysed. Distinct differences in the Aβ42 kinetic profiles were apparent and were further investigated kinetically and structurally by using thioflavin T (ThT) and transmission electron microscopy (TEM), respectively. LMW inhibitors were shown to have a differential impact on early-state aggregation. Insight provided here could direct future therapeutic design based on kinetic profiling of the process of fibril formation.     

Al2O3─SiC Composites from Aluminosilicate Precursors

Al₂O₃ and SiC composite materials have been produced from mixtures of aluminosilicates (both natural minerals and synthetic) and carbon as precursor materials. These composites are produced by heating a mixture of kaolinite (or synthetic aluminosilicates) and carbon in stoichiometric proportion above 1550°C, so that only Al₂O₃ and SiC remain as the major phases. A similar process has also been used for synthesizing other composite powders having mixtures of Al₂O₃, SiC, TiC, and ZrO₂ in different proportions (all compounds together or selective mixtures of some of them), as desired. The microstructure of hot-pressed dense compacts, produced from these powders, revealed that the SiC phase is distributed very homogeneously, even occasionally within Al₂O₃ grains on a nanosize scale. The homogeneous distribution of SiC particles within the system produced high fracture toughness of the hot-pressed material (K_IC∼ 7.0 MPa · m^1/2) and having Vicker's hardness values greater than 2000 kgf/mm².     

Digital PCR Panel for Sensitive Hematopoietic Chimerism Quantification after Allogeneic Stem Cell Transplantation

Accurate and sensitive determination of hematopoietic chimerism is a crucial diagnostic measure after allogeneic stem cell transplantation to monitor engraftment and potentially residual disease. Short tandem repeat (STR) amplification, the current “gold standard” for chimerism assessment facilitates reliable accuracy, but is hampered by its limited sensitivity (≥1%). Digital PCR (dPCR) has been shown to combine exact quantification and high reproducibility over a very wide measurement range with excellent sensitivity (routinely ≤0.1%) and thus represents a promising alternative to STR analysis. We here aimed at developing a whole panel of digital-PCR based assays for routine diagnostic. To this end, we tested suitability of 52 deletion/insertion polymorphisms (DIPs) for duplex analysis in combination with either a reference gene or a Y-chromosome specific PCR. Twenty-nine DIPs with high power of discrimination and good performance were identified, optimized and technically validated. We tested the newly established assays on retrospective patient samples that were in parallel also measured by STR amplification and found excellent correlation. Finally, a screening plate for initial genotyping with DIP-specific duplex dPCR assays was designed for convenient assay selection. In conclusion, we have established a comprehensive dPCR system for precise and high-sensitivity measurement of hematopoietic chimerism, which should be highly useful for clinical routine diagnostics.     

Aryl Bis‐Sulfonamide Inhibitors of IspF from Arabidopsis thaliana and Plasmodium falciparum

2‐Methylerythritol 2,4‐cyclodiphosphate synthase (IspF) is an essential enzyme for the biosynthesis of isoprenoid precursors in plants and many human pathogens. The protein is an attractive target for the development of anti‐infectives and herbicides. Using a photometric assay, a screen of 40 000 compounds on IspF from Arabidopsis thaliana afforded symmetrical aryl bis‐sulfonamides that inhibit IspF from A. thaliana (AtIspF) and Plasmodium falciparum (PfIspF) with IC₅₀ values in the micromolar range. The ortho‐bis‐sulfonamide structural motif is essential for inhibitory activity. The best derivatives obtained by parallel synthesis showed IC₅₀ values of 1.4 μm against PfIspF and 240 nm against AtIspF. Substantial herbicidal activity was observed at a dose of 2 kg ha^?1. Molecular modeling studies served as the basis for an in silico search targeted at the discovery of novel, non‐symmetrical sulfonamide IspF inhibitors. The designed compounds were found to exhibit inhibitory activities in the double‐digit micromolar IC₅₀ range.     

π-ELECTRON CONTENTS OF RINGS IN THE DOUBLE-HEXAGONAL-CHAIN HOMOLOGOUS SERIES (PYRENE,ANTHANTHRENE AND OTHER ACENOACENES)

Abstract

Recently three methods for calculating the π-electron content of rings of benzenoid hydrocarbons were put forward: one based on the consideration of Kekuléstructural formulas, and the other two based on an analogous treatment of the Clar aromatic sextet formulas. These three methods are applied to the homologous series consisting of two condensed acene chains (whose first members are pyrene, anthanthrene, peri-naphthacenonaphthacene, …), leading to basically identical results. In contrast to acenes (in which the partition of π-electrons into rings is uniform), in the double-hexagonal-chain species the partition of π-electrons is highly non-uniform. The electron content monotonically decreases, in opposite directions, along the two acene chains, being maximal in the least annelated rings. Some other generally valid regularities in the π-electron properties of the double–hexagonal–chain benzenoids are also pointed out.     

Optimizing the Kinetics and Thermodynamics of DNA i‐Motif Folding

Under slightly acidic conditions, single cytidine‐rich DNA strands can form four‐stranded structures called i‐motifs. The stability of the i‐motif structure is based on the intercalation of hemiprotonated C–C⁺ base pairs. In addition, the stability of these structures is influenced by pH, temperature, salt concentration, number of cytidines per C‐rich stretch, and length of sequence; it also depends on the nucleotides in the connecting loop regions. Here, we investigated the influence of the loop nucleotides on i‐motif stability, structure, and kinetics of folding, in five structures with the same loop‐size but different adenosine and thymidine residues within the loop. The stabilities of the i‐motif structures were determined by CD melting, and structure and kinetics of folding were studied by static and time‐resolved NMR experiments.     

The effects of geometry on skin penetration and failure of polymer microneedles

Microneedles are small-scale devices that may be used for drug delivery and biosensing. In this study, the forces required for mechanical failure, the modes of mechanical failure, as well as the mechanisms for microneedle penetration into porcine skin were examined. Microneedles produced from the acrylate-based polymer e-Shell 200 using an indirect rapid prototyping approach involving two-photon polymerization and poly(dimethylsiloxane) micromolding were found to possess sufficient strength for penetration of porcine skin. The failure forces were an order of magnitude greater than the forces necessary for full insertion into the skin. Bending was the most common form of failure; an increasing aspect ratio and a decreasing tip diameter were associated with lower failure forces. Video captured during skin penetration revealed that microneedle penetration into the skin occurred by means of a series of insertions and not by means of a single insertion event. Images obtained during and after skin penetration confirmed microneedle penetration of skin as well as transdermal delivery of lucifer yellow dye. These findings shed insight into the mechanisms of microneedle penetration and failure, facilitating design improvements for polymer microneedles.     

The Potential Role of Lycopene for the Prevention and Therapy of Prostate Cancer: From Molecular Mechanisms to Clinical Evidence

Lycopene is a phytochemical that belongs to a group of pigments known as carotenoids. It is red, lipophilic and naturally occurring in many fruits and vegetables, with tomatoes and tomato-based products containing the highest concentrations of bioavailable lycopene. Several epidemiological studies have linked increased lycopene consumption with decreased prostate cancer risk. These findings are supported by in vitro and in vivo experiments showing that lycopene not only enhances the antioxidant response of prostate cells, but that it is even able to inhibit proliferation, induce apoptosis and decrease the metastatic capacity of prostate cancer cells. However, there is still no clearly proven clinical evidence supporting the use of lycopene in the prevention or treatment of prostate cancer, due to the only limited number of published randomized clinical trials and the varying quality of existing studies. The scope of this article is to discuss the potential impact of lycopene on prostate cancer by giving an overview about its molecular mechanisms and clinical effects.