Atomic and Electronic Structure in MgO-SiO2.

Understanding disordered structure is difficult due to insufficient information in experimental data. Here, we overcome this issue by using a combination of diffraction and simulation to investigate oxygen packing and network topology in glassy (g-) and liquid (l-) MgO-SiO2 based on a comparison with the crystalline topology. We find that packing of oxygen atoms in Mg2SiO4 is larger than that in MgSiO3, and that of the glasses is larger than that of the liquids. Moreover, topological analysis suggests that topological similarity between crystalline (c)- and g-(l-) Mg2SiO4 is the signature of low glass-forming ability (GFA), and high GFA g-(l-) MgSiO3 shows a unique glass topology, which is different from c-MgSiO3. We also find that the lowest unoccupied molecular orbital (LUMO) is a free electron-like state at a void site of magnesium atom arising from decreased oxygen coordination, which is far away from crystalline oxides in which LUMO is occupied by oxygen's 3s orbital state in g- and l-MgO-SiO2, suggesting that electronic structure does not play an important role to determine GFA. We finally concluded the GFA of MgO-SiO2 binary is dominated by the atomic structure in terms of network topology.

Resveratrol exhibits diverse anti-cancer activities through epigenetic regulation of E-cadherin and p21 in triple-negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) has an aggressive phenotype and poor outcome, however no specific targeted therapy has been established for TNBC lacking germline BRCA1/2 pathogenic variants. To develop a novel therapeutic strategy, we explored the potential of resveratrol (RSV) for TNBC treatment. METHODS: We investigated the effects of RSV on malignant phenotypes of TNBC cells as well as on apoptosis induced by ABT263, a specific inhibitor of BCL-2 and BCL-xL, using morphological observation, migration assay, ß-galactosidase staining, and Hoechst staining. To elucidate the underlying mechanisms of RSV-mediated effects, expression levels and histone acetylation levels of cadherin 1 (CDH1, E-cadherin) and cyclin dependent kinase inhibitor 1A (CDKN1A, p21) were determined by RT-qPCR, western blotting, and chromatin immunoprecipitation. Furthermore, knockdown analysis was conducted to evaluate the involvement of E-cadherin and/or p21 in RSV potentiation on cytotoxic activity of ABT263. RESULTS: RSV treatment induced epithelial-like cellular morphology and suppressed the migration capacity in MDA-MB-231 and BT-549-Luc TNBC cells. ß-galactosidase-positive cells were increased after RSV treatment, indicating the induction of cellular senescence, in MDA-MB-231 cells but not in BT-549-Luc cells. RSV increased the expression and histone acetylation of CDH1 and CDKN1A in both cells. Interestingly, pre-treatment with RSV enhanced the induction of apoptosis in the ABT263-treated MDA-MB-231 and BT-549-Luc cells, and knockdown of CDKN1A decreased ABT263-induced apoptosis in RSV-treated MDA-MB-231 cells. CONCLUSIONS: RSV represses the metastatic capacity and enhances the cytotoxic activity of ABT263 in TNBC cells. Our results suggested that RSV can potentially be used as a repressor of metastasis or a sensitizer to ABT263 for TNBC treatment via up-regulation of CDH1 and CDKN1A through epigenetic mechanisms.

Structural Origin of Additional Infrared Transparency and Enhanced Glass-Forming Ability in Rare-Earth-Rich Borate Glasses without B-O Networks.

R2O3-B2O3 binary glasses (R denotes rare-earth elements or Y) were fabricated in a very wide composition region using a levitation technique. The maximum R2O3 content of light rare-earth compounds reached 63 mol % and decreased with a decrease in the ionic radius of R3+. The thermal, optical, vibrational, and structural properties were investigated, particularly for 50R2O3-50B2O3 glasses. The glass transition temperature increased with a decrease in the ionic radius of R3+, while the thermal stability was not affected by the glass composition. The packing density increased with a decrease in the ionic radius of R3+ due to lanthanoid contraction. Raman scattering and Fourier transform infrared spectra revealed that, in the rare-earth-rich glasses, no conventional three-dimensional networks consisting of corner-sharing BOn (n = 3 or 4) units existed because all B atoms were formed as isolated BO3 units. The simple environment around B atoms in the glasses led to additional IR transmittance regions, irrespective of the kinds of R. The total correlation functions obtained from high-energy X-ray diffraction measurements were analyzed using the pair-function method and compared with those of various RBO3 crystalline phases. It was suggested that the local structure around R resembles the ν-NdBO3-type crystal structure, and the O coordination number of R ranged from 6.5 to 7.7, smaller than that of the crystalline phase. The glass-forming ability depending on R was discussed based on the structural similarities between the melt, glass, and crystalline phases.

High refractive index La-rich lanthanum borate glasses composed of isolated BO3 units.

La2O3-B2O3 binary glasses were prepared by containerless processing using a levitation technique. The bulk glass-forming region in a B-rich composition was extended compared to that using conventional melt-quench techniques. Furthermore, additional glass formation was realized in an La-rich composition. The glass transition temperature and crystallization temperature of La-rich glasses are much higher than those of B-rich glasses. Both B- and La-rich glasses were colorless and transparent and had a high refractive index with low wavelength dispersion in the visible region. With the increase of the La2O3 content, the optical absorption edge in the ultraviolet (UV) region shifts to a long wavelength. An additional infrared (IR) transmittance window was observed in La-rich glasses, indicating that the La-rich borate glasses are expected to be used in optical components in a wide wavelength region. Local structural analyses using 11B magic-angle spinning (MAS) nuclear magnetic resonance (NMR) and Raman scattering spectra revealed that every B-atom in La-rich glasses formed a planar trigonal BO3 unit and these BO3 units were entirely isolated. The evident difference from B-rich glasses where B-atoms formed a complex network structure with BO3 and BO4 units caused the characteristic physical and optical properties of La-rich glasses.

Single CpG site methylation controls estrogen receptor gene transcription and correlates with hormone therapy resistance.

Hormone therapy is the most effective treatment for patients with estrogen receptor α-positive breast cancers. However, although resistance occurs during treatment in some cases and often reflects changed estrogen receptor α status, the relationship between changes in estrogen receptor α expression and resistance to therapy are poorly understood. In this study, we identified a mechanism for altered estrogen receptor α expression during disease progression and acquired hormone therapy resistance in aromatase inhibitor-resistant breast cancer cell lines. Subsequently, we investigated promoter switching and DNA methylation status of the estrogen receptor α promoter, and found marked changes of methylation at a single CpG site (CpG4) in resistant cells. In addition, luciferase reporter assays showed reduced transcriptional activity from this methylated CpG site. This CpG region was also completely conserved among species, suggesting that it acts as a methylation-sensitive Ets-2 transcription factor binding site, as confirmed using chromatin immunoprecipitation assays. In estrogen receptor α-positive tumors, CpG4 methylation levels were inversely correlated with estrogen receptor α expression status, suggesting that single CpG site plays an important role in the regulation of estrogen receptor α transcription.

Crystal structures of the S6K1 kinase domain in complexes with inhibitors.

Ribosomal protein S6 kinase 1 (S6K1) is a serine/threonine protein kinase that plays an important role in the PIK3/mTOR signaling pathway, and is implicated in diseases including diabetes, obesity, and cancer. The crystal structures of the S6K1 kinase domain in complexes with staurosporine and the S6K1-specific inhibitor PF-4708671 have been reported. In the present study, five compounds (F108, F109, F176, F177, and F179) were newly identified by in silico screening of a chemical library and kinase assay. The crystal structures of the five inhibitors in complexes with the S6K1 kinase domain were determined at resolutions between 1.85 and 2.10 Å. All of the inhibitors bound to the ATP binding site, lying along the P-loop, while the activation loop stayed in the inactive form. Compound F179, with a carbonyl group in the middle of the molecule, altered the αC helix conformation by interacting with the invariant Lys123. Compounds F176 and F177 bound slightly distant from the hinge region, and their sulfoamide groups formed polar interactions with the protein. The structural features required for the specific binding of inhibitors are discussed.

Prediction of ligand-induced structural polymorphism of receptor interaction sites using machine learning.

Protein functions are closely related to their three-dimensional structures. Various degrees of conformational changes in the main and side chains occur when binding with other molecules, such as small ligands or proteins. The ligand-induced structural polymorphism of proteins is also referred to as "induced-fit", and it plays an important role in the recognition of a particular class of ligands as well as in signal transduction. We have developed new prediction models that discriminate conformationally fluctuant residues caused by ligand-binding. The training and test data sets were obtained from the Protein Data Bank. The induced-fit residues were judged based on the Z values of the Cα atom distances in each protein cluster. Moreover, we introduced various descriptors, such as the number of residues, accessible surface area (ASA), depth of the residue, and position-specific scoring matrix (PSSM), which were obtained from the 2D- or 3D-structural information for the protein. After the optimization of the parameters by 5-fold cross validation, the best prediction model was applied to some well-known induced-fit target proteins to verify its effectiveness. Especially in the validation for the DFG motif of a protein kinase family, we succeeded in the prediction of the DFG-out possibility from only the DFG-in conformation of each kinase structure.

Application of support vector machine to three-dimensional shape-based virtual screening using comprehensive three-dimensional molecular shape overlay with known inhibitors.

In this study, machine learning using support vector machine was combined with three-dimensional (3D) molecular shape overlay, to improve the screening efficiency. Since the 3D molecular shape overlay does not use fingerprints or descriptors to compare two compounds, unlike 2D similarity methods, the application of machine learning to a 3D shape-based method has not been extensively investigated. The 3D similarity profile of a compound is defined as the array of 3D shape similarities with multiple known active compounds of the target protein and is used as the explanatory variable of support vector machine. As the measures of 3D shape similarity for our new prediction models, the prediction performances of the 3D shape similarity metrics implemented in ROCS, such as ShapeTanimoto and ScaledColor, were validated, using the known inhibitors of 15 target proteins derived from the ChEMBL database. The learning models based on the 3D similarity profiles stably outperformed the original ROCS when more than 10 known inhibitors were available as the queries. The results demonstrated the advantages of combining machine learning with the 3D similarity profile to process the 3D shape information of plural active compounds.

Pyrrole-imidazole hairpin polyamides with high affinity at 5'-CGCG-3' DNA sequence; influence of cytosine methylation on binding.

To investigate the binding of 5'-CpG-3' sequences by small molecules, two pyrrole (Py)-imidazole (Im) hairpin polyamides, PyImPyIm-gamma-PyImPyIm-beta-Dp (1) and PyIm-beta-Im-gamma-PyIm-beta-Im-beta-Dp (2), which recognize the sequence 5'-CGCG-3', were synthesized. The binding affinities of the 5'-CGCG-3' sequence to the Py-Im hairpin polyamides were measured by surface plasmon resonance (SPR) analysis. SPR data revealed that dissociation equilibrium constants (K(d)) of polyamides 1 and 2 were 1.1 (+/- 0.3) x 10(-6) M and 1.7 (+/- 0.4) x 10(-8) M, respectively. Polyamide 2 possesses great binding affinity for this sequence, 65-fold higher than polyamide 1. Moreover, when all cytosines in 5'-CpGpCpG-3' were replaced with 5-methylcytosines ((m)Cs), the K(d) value of polyamide 2 increased to 5.8 (+/- 0.7) x 10(-9) (M), which indicated about 3-fold higher binding than the unmethylated 5'-CGCG-3' sequence. These results suggest that polyamide 2 would be suitable to target CpG-rich sequences in the genome.

Sequence-specific alkylation by Y-shaped and tandem hairpin pyrrole-imidazole polyamides.

To extend the target DNA sequence length of the hairpin pyrrole-imidazole (Py-Im) polyamide 1, we designed and synthesized Y-shaped and tandem hairpin Py-Im polyamides 2 and 3, which possess 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) as DNA-alkylating moieties. High-resolution denaturing polyacrylamide gel electrophoresis by using 5'-Texas-Red-labeled 465 base pair (bp) DNA fragments revealed that conjugates 2 and 3 alkylated the adenine of the target DNA sequences at nanomolar concentrations. Conjugate 2 alkylated adenine N3 at the 3' end of two 8 bp match sequences, 5'-AATAACCA-3' (site A) and 5'-AAATTCCA-3' (site C), while conjugate 3 recognized one 10 bp match sequence, 5'-AGAATAACCA-3' (site A) in the 465 bp DNA fragments. These results demonstrate that seco-CBI conjugates of Y-shaped and tandem hairpin polyamides have extended their target alkylation sequences.

Inducing the replacement of PNA in DNA.PNA duplexes by DNA.

The uncharged DNA-analogue peptide nucleic acid (PNA) can invade into dsDNA by displacing the non-complementary DNA strand. The formed strand displacement complexes can create a sterical hindrance to block access of enzymes such as nucleases and polymerases. Due to the high stability of DNA.PNA duplexes it is usually not possible to displace the PNA strand by ssDNA or ssRNA. We herein report that the polycationic, comb-type copolymer alphaPLL-g-Dex can induce such a replacement of PNA in DNA.PNA duplexes by ssDNA. The influence of the copolymer on strand exchange highly depends on the nature of the oligonucleotides. Acceleration has only been observed when both the starting duplex and the single-stranded exchanger strand were negatively charged. The presented approach should allow the withdrawal of PNA induced sterical hindrance of DNA by rehybridisation with ssDNA.

Requirement of beta-alanine components in sequence-specific DNA alkylation by pyrrole-imidazole conjugates with seven-base pair recognition.

To investigate the effect of incorporation of beta-alanine in alkylating N-methylpyrrole (Py)-N-methylimidazole (Im) polyamide, seco-CBI conjugates 2-8 were synthesized by an Fmoc solid-phase method and subsequent coupling with an alkylating moiety. DNA-alkylating activities of conjugates 2-8 were evaluated by high-resolution denaturing gel electrophoresis with 202-base pair (bp) DNA fragments. Alkylation by conjugates 2 and 3, which have antiparallel pairings of beta-alanine (beta) opposite beta (beta/beta) and Py/beta, occurred mainly at the adenine (A) of the matching sequences, 5'-AGCTCCA-3' (site 1) and 5'-AGCACCA-3' (site 3). However, conjugate 4, with beta/Py, did not show any DNA-alkylating activities. Similarly, conjugate 5, which possessed a Py/Py pair, weakly alkylated the matching sites at micromolar concentrations. Conjugates 6 and 7, which possessed beta/beta and Py/beta pairs, respectively, alkylated at the A of the matching sequences, 5'-ACTACCA-3' (site 2) and 5'-ACAACCA-3' (site 4). In contrast, conjugated 8, with a Py/Py pair, showed lower activity and less alkylated DNA at sites 2 and 4 with mismatched alkylation at site 1 at a higher concentration than that of 6 and 7. These results demonstrate that incorporation of beta-alanine is required for the sequence-specific alkylation by seco-CBI Py-Im conjugates with a seven-base pair sequence.

Synthesis and biological properties of pyrrole-imidazole polyamide conjugates.

We have developed a series of conjugates between pyrrole (Py)-imidazole (Im) polyamides and 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H benz[e]indole (seco-CBI) with an indole linker. High resolution polyacrylamide gel electrophoresis revealed that these conjugates alkylated DNA at predetermined sequences. Then, we demonstrated that conjugates 1 and 2 have DNA alkylation activities at double stranded human telomere sequence and potent cytotoxicities in cancer cell lines. In addition, we showed that conjugate 3 alkylates DNA with ten-base-pair recognition sequence in the presence of partner polyamide 4, which suggested alkylation through 3-4 heterodimer formation.

Sequence-specific alkylation by a tandem motif of pyrrole-imidazole CBI conjugate.

We have developed various types of sequence-specific alkylating agents by conjugation of Py-Im polyamides and alkylating moieties 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) with an indole linker. In order to extend the length of target DNA sequence of the hairpin Py-Im polyamide 1, we designed and synthesized Y-shaped and tandem hairpin Py-Im polyamides 2 and 3. High-resolution denaturing polyacrylamide gel electrophoresis using 5'-Texas Red-labeled 465-bp DNA fragments revealed that conjugates 2 and 3 alkylated the adenine of target DNA sequences at nanomolar concentrations. Furthermore, evaluation in human cancer cell lines revealed that these Py-Im polyamides 2 and 3 have strong cytotoxicities.

Detection of CAG repeat DNA sequences by pyrene-functionalized pyrrole-imidazole polyamides.

Five N-methylpyrrole-N-methylimidazole (Py-Im) polyamides possessing a fluorescent pyrene were synthesized by Fmoc solid-phase synthesis using Py/Im monomers and pyrenylbutyl-pyrrole monomer compound 9. The steady state fluorescence of conjugates 1-5 was examined in the presence and absence of (CAG)(12)-containing oligodeoxynucleotides (ODNs) 1 and 2. Of the conjugates, conjugate 1 showed no background emission around 470 nm in the absence of ODNs, and a clear increase of emission at 475 nm was observed upon addition of ODNs 1 and 2. The emission of conjugate 1 at 475 nm increased linearly with the concentration of ODN and the number of CAG repeats. The results indicate that conjugate 1 efficiently forms a pyrene excimer upon binding in the minor groove of DNA.

DNA alkylation by pyrrole-imidazole seco-CBI conjugates with an indole linker: sequence-specific DNA alkylation with 10-base-pair recognition through heterodimer formation.

The sequence-specific DNA alkylation by conjugates 4 and 5, which consist of N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides and 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) linked with an indole linker, was investigated in the absence or presence of partner Py-Im polyamide 6. High-resolution denaturing polyacrylamide gel electrophoresis revealed that conjugate 4 alkylates DNA at the sequences 5'-(A/T)GCCTA-3' through hairpin formation, and alkylates 5'-GGAAAGAAAA-3' through an extended binding mode. However, in the presence of partner Py-Im polyamide 6, conjugate 4 alkylates DNA at a completely different sequence, 5'-AGGTTGTCCA-3'. Alkylation of 4 in the presence of 6 was effectively inhibited by a competitor 7. Surface plasmon resonance (SPR) results indicated that conjugate 4 does not bind to 5'-AGGTTGTCCA-3', whereas 6 binds tightly to this sequence. The results suggest that alkylation proceeds through heterodimer formation, indicating that this is a general way to expand the recognition sequence for DNA alkylation by Py-Im seco-CBI conjugates.

The biological impact of sequence-specific DNA alkylation by pyrrole-imidazole polyamides.

We have developed a series of novel DNA alkylating polyamides possessing indole linkers. Investigations using high-resolution gel electrophoresis revealed that the indole linked Py-Im polyamide alkylated at A of a targeted nine base pair matching sequence. Evaluation in human cancer cell lines revealed that the indole linked Py-Im polyamides have strong cytotoxicities. Furthermore, we showed that alkylation of the template strand of the coding region by these polyamides causes effective gene silencing.

Synthesis and evaluation of sequence-specific DNA alkylating agents: effect of alkylation subunits.

We have demonstrated that hairpin pyrrole (Py)- imidazole (Im) polyamide-CBI conjugates selectively alkylate predetermined sequences. In this study, we investigated the effect of alkylation subunits, for example conjugates 1-4 with three types of DNA alkylating units, and Py-Im polyamides with indole linker. Conjugate 3 and 4 selectively alkylated the predetermined sequences as described previously, while conjugates 1 and 2 alkylate at mismatched sites.

Molecular design of DNA alkylating pyrrole-imidazole polyamides with longer recognition sequence.

The sequence-specificity, and DNA alkylating activity of the conjugate 1, which consists of N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides, 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) with indole linker, were investigated in the absence or presence of partner Py-Im polyamide 2. High-resolution denaturing polyacrylamide gel electrophoresis showed that the specificity of DNA alkylation by 1 modulated in the presence of partner 2. We found that sequence-specific DNA alkylation by 1 and 2 with 10 base pair (bp) match recognition sequence through heterodimer formation. This result indicates one possibility of DNA alkylation with longer recognition sequence by different two molecules.

Sequence-specific alkylation of double-strand human telomere repeat sequence by pyrrole-imidazole polyamides with indole linkers.

We designed and synthesized pyrrole (Py)-imidazole (Im) hairpin polyamide 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) conjugates 1 and 2, which target both strands of the double-stranded region of the human telomere repeat sequences, 5'-d(TTAGGG)(n)-3'/5'-d(CCCTAA)(n)-3'. High-resolution denaturing polyacrylamide gel electrophoresis demonstrated that conjugates 1 and 2 alkylated DNA at the 3' A of 5'-ACCCTA-3' and 5'-AGGGTTA-3', respectively. Cytotoxicities of conjugates 1 and 2 were evaluated using 39 human cancer cell lines; averages of log IC(50) values for conjugates 1 and 2 were -6.96 (110 nM) and -7.24 (57.5 nM), respectively. Conjugates 1 and 2 have potential as antitumor drugs capable of targeting telomere repeat sequence.