Production of monoclonal antibodies against hop-derived (Humulus lupulus L.) prenylflavonoids and the development of immunoassays

Monoclonal antibodies against the hop-derived prenylated chalcone xanthohumol (X) and the prenylated flavonoids isoxanthohumol (IX) and 8-prenylnaringenin (8-PN) were developed. Carboxylic acid haptens of X, IX and 8-PN were synthesized by linking a spacer to their C4′-OH group followed by subsequent coupling to bovine serum albumin (BSA) to form conjugates that were employed as immunogens in BALB/c mice to raise antibodies. The monoclonal antibodies that were secreted from the established hybridoma cell lines proved, in cross-reactivity studies, to possess highly specific binding capacities in an optimized competitive indirect ELISA. The immunoassays make use of immunogen-coated microtiterplates and a peroxidase-labeled anti-mouse IgG₁ secondary antibody with ABTS as a chromogenic substrate. For X the IC₅₀ value derived from the standard curve was 62.91 ng mL⁻¹, and for both IX and 8-PN 37.15 ng mL⁻¹. The assay was validated for the quantitative analysis of X, IX and 8-PN in urine and serum. A simple sample pretreatment procedure using a diethyl ether extraction was optimized and the recoveries and matrix effects were assessed. The validity of the established assay was tested and mean inter- and intra-assay variations in urine were 2.32% and 1.91%, respectively for X, 6.24% and 2.39%, respectively for IX and 7.18% and 0.74%, respectively for 8-PN. In serum, the mean inter- and intra-assay variations were 8.90% and 1.37%, respectively for X, 6.13% and 1.57%, respectively for IX and 6.13% and 2.43%, respectively for 8-PN. Furthermore, the method demonstrated excellent accuracy and significant correlation with measurements by an established and validated HPLC-MS method.     

Application of a ligand-based theoretical approach to derive conversion paths and ligand conformations in CYP11B2-mediated aldosterone formation

The biosynthesis of the mineralocorticoid hormone aldosterone involves a multistep hydroxylation of 11-deoxycorticosterone at the 11- and 18-positions, resulting in the formation of corticosterone and 18-hydroxycorticosterone, the final precursor of aldosterone. Two members of the cytochrome P450 11B family, CYP11B1 and CYP11B2, are known to catalyze these 11- and 18-hydroxylations, however, only CYP11B2 can oxidize 18-hydroxycorticosterone to aldosterone. It is unknown what sequence of hydroxylations leads to the formation of 18-hydroxycorticosterone. In this study we have investigated which of the possible conversion paths towards formation of 18-hydroxycorticosterone and aldosterone are most likely from the ligand perspective. Therefore, we combined quantum mechanical investigations on the steroid conformations of 11-deoxycorticosterone and its ensuing reaction intermediates with Fukui indices calculations to predict the reactivity of their carbon atoms for an attack by the iron-oxygen species. Both F(-) and F(0) were calculated to account for different mechanisms of substrate conversion. We show which particular initial conformations of 11-deoxycorticosterone and which conversion paths are likely to result in the successful synthesis of aldosterone, and thereby may be representative for the mechanism of aldosterone biosynthesis by CYP11B2. Moreover, we found that the most likely path for aldosterone synthesis coincides with the substrate conformation proposed in an earlier publication. To summarize, we show that on a theoretical and strictly ligand-directed basis only a limited number of reaction paths in the conversion of 11-deoxycorticosterone to aldosterone is possible. Despite its theoretical nature, this knowledge may help to understand the catalytic function of CYP11B1 and CYP11B2.     

Ion mobility spectrometry of talarozole, a new azole drug, in cleaning quality control

Ion mobility spectrometry (IMS) gains increased pharmaceutical interest as an analytical technique for the verification of equipment cleaning. Using a fractional factorial design, we developed an IMS method for talarozole, which represents a new generation of retinoic acid metabolism blocking agents (RAMBA) that can be used for the treatment of different dermatological diseases, such as psoriasis and acne. Using a Smiths Detection Ionscan-LS and the optimal IMS settings obtained, talarozole showed a drift time of 16.648 ms, corresponding to a reduced ion mobility K₀ of 1.072 cm²V^?1s^?1. Total analysis times below 1 min were achieved. Talarozole was well separated in the plasmagram from other azole compounds and the limit of detection was found to be 43 ng/ml. Swab samples collected from steel and glass plates were successfully analyzed, thereby showing that IMS is indeed a suitable technique for the quantitative analysis of talarozole in cleaning quality control.     

Potential energy surface and matrix isolation FT-IR study of isoleucine

The conformational landscape of isoleucine was investigated by a theoretical DFT and MP2 study. This investigation has revealed new important conformations in comparison to a previous study. Five conformations have been predicted with an abundance larger than 5% at both levels of theory. Among these, three different types of amino acid backbone were present, each characterized by a typical intramolecular H-bond. Due to the H-bonding, the frequencies of the involved groups are strongly shifted, and these modes were probably observed with FT-IR spectroscopy in an Ar matrix. The experimental abundances could be estimated and were in good accordance with the MP2 stabilities, whereas the accordance with the DFT abundances was poor. In contrast to this, the correspondence of the DFT and the experimental frequencies was excellent, as demonstrated by the mean frequency deviation of only 4.7 cm(-1).     

Excitation energy shuttling in oligothiophene-diketopyrrolopyrrole-fullerene triads

The photophysical properties of a thiophene-diketopyrrolopyrrole oligomer linked to two fullerene units via alkyl linkers of different lengths have been investigated in solution. The molecules exhibit excitation energy shuttling between the singlet and triplet photoexcited states. Photoexcitation of the oligomer is followed by singlet energy transfer to the fullerene, intersystem crossing to the triplet state, and then triplet energy transfer back to the oligomer. Competing electron transfer reactions, followed by recombination to the triplet state, are energetically possible and cannot be ruled out but were not observed and seem to have a small contribution in solution.     

Verifying the attenuation of earplugs in situ: method validation using artificial head and numerical simulations

The use of in situ measurements of hearing protectors' (HPD's) attenuation following the microphone in real ear (MIRE) protocol is increasing. The attenuation is hereby calculated from the difference in sound levels outside the ear and inside the ear canal behind the HPD. Custom-made earplugs have been designed with an inner bore that allows inserting a miniature microphone. A thorough understanding of the difference, henceforth called transfer function, between the sound pressure of interest at the eardrum and the one measured at the inner bore of the HPD is indispensable for optimizing the MIRE technique and extending its field of application. This issue was addressed by measurements on a head-and-torso-simulator and finite difference time domain numerical simulations of the outer ear canal occluded by an earplug. Both approaches are in good agreement and reveal a clear distinction between the sound pressure at the MIRE microphone and at eardrum, but the measured transfer functions appear to be stable and reproducible. Moreover, the most striking features of the transfer functions can be traced down to the geometrical and morphological characteristics of the earplug and ear canal.     

The η−η' mixing angle from duality and quark model

From simple quark model and duality arguments the η?η' mixing angle is predicted to be $\text{θ =}\text{arctan}\text{(}\text{?1}\text{2}\text{√2) ≈ ?19.5°}$. The available experimental data seem to support such a result.     

Easy and efficient anion alkylations in solid-liquid PTC conditions

Alkylation of anionic nucleophiles such as potassium acetate or potassium indole can be achieved in good yields without solvent either in the presence of NBu₄Br and small amounts of TiO₂ or in the presence of Aliquat 336 (Oct₃MeN⁺Cl^?).     

Alkylation of anions on solid inorganic supports : an interpretation of the function of the supports

In the anionic alkylation (CH₃ CO₂K + n C₈ H₁₇ Br) on solid inorganic supports, silica impregnated with cationic surfactant appears to be as effective as alumina, while silica bearing ammonium groups covalently bonded to the support (SPHEROSIL QMA) exhibits increased effectiveness. An interpretation based upon the superficial charge of the solid surfaces is proposed.     

Ultrasonic cleaning of 3D printed objects and Cleaning Challenge Devices

We report our experiences in the evaluation of ultrasonic cleaning processes of objects made with additive manufacturing techniques, specifically three-dimensional (3D) printers. These objects need to be cleaned of support material added during the printing process. The support material can be removed by dissolution in liquids with or without ultrasonic cavitation.