Monitoring the site-specific incorporation of dual fluorophore-quencher base analogues for target DNA detection by an unnatural base pair system

We developed intramolecular dual fluorophore-quencher base analogues for site-specific incorporation into DNA by an unnatural base pair replication system. An unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) exhibits high fidelity in PCR amplification, and the 2-nitropyrrole moiety of Px acts as a quencher. Deoxyribonucleoside triphosphates of Px linked with a fluorophore (Cy3, Cy5 or FAM) were chemically synthesized, and the fluorescent properties and the enzymatic incorporation of the fluorophore-linked dPxTPs into DNA were examined in PCR amplification. The fluorophore-linked dPxTPs were site-specifically incorporated by PCR into DNA, opposite Ds in templates, with high selectivity. Furthermore, we found that the fluorescence of the triphosphates was partially quenched, but increased upon their incorporation into DNA. These dual fluorophore-quencher base analogues would be useful for site-specific DNA labeling and for monitoring the amplification products of target nucleic acid molecules with a specific sequence. We have demonstrated the utility of the fluorophore-linked Px substrates and the Ds-Px pairing in real-time quantitative PCR for target DNA molecule detection.     

Syntheses, characterization, and photochemical properties of amidate-bridged Pt(bpy) dimers tethered to Ru(bpy)3(2+) derivatives

Amidate-bridged diplatinum(II) entities [Pt(2)(bpy)(2)(μ-amidato)(2)](2+) (amidate = pivalamidate and/or benzamidate; bpy = 2,2'-bipyridine) were covalently linked to one or two Ru(bpy)(3)(2+)-type derivatives. An amide group was introduced at the periphery of Ru(bpy)(3)(2+) derivatives to give metalloamide precursors [Ru(bpy)(2)(BnH)](2+) (abbreviated as RuBnH, n = 1 and 2), where deprotonation of amide BnH affords the corresponding amidate Bn, B1H = 4-(4-carbamoylphenyl)-2,2'-bipyridine, and B2H = ethyl 4'-[N-(4-carbamoylphenyl)carbamoyl]-2,2'-bipyridine-4-carboxylate. From a 1:1:1 reaction of [Pt(2)(bpy)(2)(μ-OH)(2)](NO(3))(2), RuBnH, and pivalamide, trinuclear complexes [Pt(2)(bpy)(2)(μ-RuBn)(μ-pivalamidato)](4+) (abbreviated as RuBn-Pt(2)) were isolated and characterized. Tetranuclear complexes [Pt(2)(bpy)(2)(μ-RuBn)(2)](6+) (abbreviated as (RuBn)(2)-Pt(2)) were separately prepared and characterized in detail. The quenching of the triplet excited state of the Ru(bpy)(3)(2+) derivative (i.e., Ru*(bpy)(3)(2+)) upon tethering the Pt(2)(bpy)(2)(μ-amidato)(2)(2+) moiety is strongly enhanced in RuB1-Pt(2) and (RuB1)(2)-Pt(2), while it is only slightly enhanced in RuB2-Pt(2) and (RuB2)(2)-Pt(2). These are partly explained by the driving forces for the electron transfer from the Ru*(bpy)(3)(2+) moiety to the Pt(2)(bpy)(2)(μ-amidato)(2)(2+) moiety (ΔG°(ET)); the ΔG°(ET) values for RuB1-Pt(2), (RuB1)(2)-Pt(2), RuB2-Pt(2), and (RuB2)(2)-Pt(2) are estimated as -0.01, 0.00, +0.22, and +0.28 eV, respectively. The considerable difference in the photochemical properties of the B1- and B2-bridged systems were further examined based on the emission decay and transient absorption measurements, which gave results consistent with the above conclusions.     

Synthesis of polyfunctional allenes by successive copper-mediated substitutions

Readily available 1,1-dichloro-2-alkynes are versatile starting materials for the synthesis of polyfunctionalized allenes. They can be prepared from commercially available terminal alkynes in a two-step procedure. The Cu-mediated reaction of several 1,1-propargylic derivatives with functionalized alkyl, benzylic, or allylic zinc reagents proceeds exclusively with S(N)2' selectivity and allows a rapid and efficient synthesis of functionalized chloroallenes. These chloroallenes undergo a novel Cu(I) -catalyzed substitution reaction with functionalized arylmagnesium reagents with excellent S(N)2 selectivity to give trisubstituted polyfunctionalized allenes. The functional group tolerance is excellent and functionalities, such as cyano, keto, ester, phosphate, trifluoromethyl, and halogens, are well tolerated.     

Gaining insight into the inhibition of glycoside hydrolase family 20 exo-β-N-acetylhexosaminidases using a structural approach

One useful methodology that has been used to give insight into how chemically synthesized inhibitors bind to enzymes and the reasons underlying their potency is crystallographic studies of inhibitor-enzyme complexes. Presented here is the X-ray structural analysis of a representative family 20 exo-β-N-acetylhexosaminidase in complex with various known classes of inhibitor of these types of enzymes, which highlights how different inhibitor classes can inhibit the same enzyme. This study will aid in the future development of inhibitors of not only exo-β-N-acetylhexosaminidases but also other types of glycoside hydrolases.     

Structure Elucidation of Textile Fabrics by Fourier Transform Infrared Photoacoustic Spectroscopy

A photoacoustic detection system mainly based on a Fourier transform infrared spectrometer has been constructed and some tentative evaluations of its performances have been made. Infrared photoacoustic spectra of cotton and nylon cloths have been obtained without troublesome sample preparations which are necessary in conventional infrared spectrometry. It can be said that the Fourier transform infrared photoacoustic spectroscopy is useful for the structure elucidation of organic polymers.     

Investigation for a Drastic Change of the Core in 23O and 24O

We study the radius and energies of oxygen isotopes towards the neutron drip-line. The abrupt increase of the radius at ²³O is investigated with two different approaches; the m-scheme cluster-orbital shell model (COSM) and a simplified model approach with the core plus one and two valence neutrons. We investigate properties of the oxygen isotopes by changing the radius of the ¹⁶O-core in the m-scheme COSM approach and obtain the result that the broad structure of ¹⁶O is favored in the ²³O and ²⁴O. In the simplified model approach, we estimate the magnitude of the change of the size of ²²O in ²³O and ²⁴O.     

Experimental determination of orientations for the 17 O electric-field-gradient and chemical shielding tensors in L-alanine

We have presented a solid-state 17 O NMR study of [13C, 17 O]-L-alanine. Using the experimental results for the 13C-17 O dipolar vector and Euler angles, the absolute orientations of 17 O chemical shielding (CS) and electric-field-gradient (EFG) tensors with respect to the molecular frame can be determined for L-alanine. The present results suggest that the intermediate EFG tensor components, VYY, lie in the carboxylate plane and parallel to the C-O bond directions, while the least shielded components, delta11, and the intermediate CS tensor components, delta22, roughly lie in the molecular plane and the direction of delta22 components are approximately 38 degrees and 25 degrees off the C-O bonds for O1 and O2, respectively. These results are in reasonable agreement with those of our quantum chemical calculations reported previously.     

Dynamic Risk Prediction via a Joint Frailty-Copula Model and IPD Meta-Analysis: Building Web Applications

Clinical risk prediction formulas for cancer patients can be improved by dynamically updating the formulas by intermediate events, such as tumor progression. The increased accessibility of individual patient data (IPD) from multiple studies has motivated the development of dynamic prediction formulas accounting for between-study heterogeneity. A joint frailty-copula model for overall survival and time to tumor progression has the potential to develop a dynamic prediction formula of death from heterogenous studies. However, the process of developing, validating, and publishing the prediction formula is complex, which has not been sufficiently described in the literature. In this article, we provide a tutorial in order to build a web-based application for dynamic risk prediction for cancer patients on the basis of the R packages joint.Cox and Shiny. We demonstrate the proposed methods using a dataset of breast cancer patients from multiple clinical studies. Following this tutorial, we demonstrate how one can publish web applications available online, which can be manipulated by any user through a smartphone or personal computer. After learning this tutorial, developers acquire the ability to build an online web application using their own datasets.