Development and application of immunoaffinity column chromatography for atrazine in complex sample media

A rabbit antibody immunoaffinity (IA) column procedure was evaluated as a cleanup method for the determination of atrazine in soil, sediment, and food. Four IA columns were prepared by immobilizing a polyclonal rabbit anti-atrazine antibody solution to HiTrap Sepharose columns. Atrazine was bound to the IA columns when the loading solvents were either 100% water, 2% acetonitrile in water, or 10% methanol in phosphate buffered saline (PBS). Quantitative removal of atrazine from the IA columns was achieved with elution solvents of either 70% ethanol in water, 70% methanol in water, or 100% methanol. One control column was prepared using nonspecific rabbit IgG antibody. This control column did not retain any applied atrazine indicating atrazine did not bind indiscriminately to protein or the Sepharose support. The four IA columns showed reproducible coupling efficiency for the immobilization of the atrazine antibody and consistent binding and releasing of atrazine. The coupling efficiency (4.25 mg of antibody in 1 mL of resin bed) for the four IA columns ranged from 93 to 97% with an average of 96 ± 2% (2.1%). Recoveries of the 500, 50, and 5 ng mL⁻¹ atrazine standard solutions from the four IA columns were 107 ± 7% (6.5%), 122 ± 14% (12%), and 114 ± 9% (8.0%) respectively, based on enzyme-linked immunosorbent assay (ELISA) data. The maximum loading was approximately 700 ng of atrazine for each IA column (∼0.16 μg of atrazine per mg of antibody). The IA columns could withstand 100% methanol as the elution solvent and could be reused more than 50 times with no change in performance. The IA columns were challenged with soil, sediment, and duplicate-diet food samples and effectively removed interferences from these various matrices for subsequent gas chromatography/mass spectrometry (GC/MS) or ELISA analysis. The log-transformed ELISA and GC/MS data were significantly correlated for soil, sediment and food samples although the ELISA values were slightly higher than those obtained by GC/MS. The IA column cleanup procedure coupled with ELISA analysis could be used as an alternative effective analytical method for the determination of atrazine in complex sample media such as soil, sediment, and food samples.     

Infrared and Raman vibrational spectroscopies reveal the palette of frescos found in the medieval monastery of Karaach Teke

Vibrational spectroscopy is applied on samples obtained from the excavation area of the medieval Monastery (10th century) of Karaach-Teke in Bulgaria. The results of the corresponding study, reveal the type of materials used for the creation of the wall-paintings and give evidence of Byzantine influence, a fact that further supports the well known impact of Byzantium on the technology and thematic-aesthetic features of iconography in Bulgaria during this era. In addition, the complementarity of FTIR and micro-Raman spectroscopies in the identification of pigments is indicated.     

“Alternative medical therapy” use among singers: Prevalence and implications for the medical care of the singer

Singers are extremely conscious of health problems that affect their voices and well-being and often take an active role in seeking care for these problems. They frequently seek treatment from providers or with modalities considered “alternative” to traditional medical care. A survey of singers was completed to elucidate their attitudes and practices regarding “alternative modalities” of medical care. Frequently singers will self-medicate or take advice from people not well versed in the special needs of a professional voice user. They will fail to share this information with the physician when seeking “traditional” medical care. These practices may predispose the singer to suboptimal or even dangerous care. These results are discussed, as well as the implications for the medical physician treating the singer. The possible detrimental pharmacologic effects of “natural therapies” widely used by singers are presented, with special attention to the particular concerns for the professional singer     

Chlovalicin B,a Chlorinated Sesquiterpene Isolated from the Marine Mushroom Digitatispora marina

As part of our search for bioactive metabolites from understudied marine microorganisms, the new chlorinated metabolite chlovalicin B (1) was isolated from liquid cultures of the marine basidiomycete Digitatispora marina, which was collected and isolated from driftwood found at Vannøya, Norway. The structure of the novel compound was elucidated by spectroscopic methods including 1D and 2D NMR and analysis of HRMS data, revealing that 1 shares its molecular scaffold with a previously isolated compound, chlovalicin. This represents the first compound isolated from the Digitatispora genus, and the first reported fumagillin/ovalicin-like compound isolated from Basidiomycota. Compound 1 was evaluated for antibacterial activities against a panel of five bacteria, its ability to inhibit bacterial biofilm formation, for antifungal activity against Candida albicans, and for cytotoxic activities against malignant and non-malignant human cell lines. Compound 1 displayed weak cytotoxic activity against the human melanoma cell line A2058 (~50% survival at 50 µM). No activity was detected against biofilm formation or C. albicans at 50 µM, or against bacterial growth at 100 µM nor against the production of cytokines by the human acute monocytic leukemia cell line THP-1 at 50 µM.     

Photocycloaddition of 2-Oxopyran-3-carbonitriles to 2,3-Dimethylbut-2-ene

On acetone-sensitized irradiation in the presence of 2,3-dimethylbut-2-ene, α-cyano-α,β-unsaturated δ-lactone 5 is converted both to cyclopentapyrans resulting from stepwise addition of the alkene to the olefinic C(β)- and the nitrile C-atom of triplet excited 5 , and cyclobutapyranes, i.e., [2+2] cycloadducts. Similarly, direct irradiation of 1-benzopyran-3-carbonitrile 6 in the presence of the same alkene affords cyclobutabenzopyran 13 and cyclopentabenzopyran 14 , the latter resulting from an upper excited triplet state of 6 .     

Ochre-differentiation through micro-Raman and micro-FTIR spectroscopies: application on wall paintings at Meteora and Mount Athos, Greece

The most widely-used inorganic pigments of Byzantine and post-Byzantine hagiography are earth pigments called ochres such as, red and yellow ochres, limonite, goethite, raw and burnt sienna, caput mortuum and hematite. The present experimental work proposes a technique of differentiation that allows one to distinguish among all the different kinds of iron oxides, thereby providing a better understanding of the painting technique used on portable icons and wall paintings. The ratios between the main spectroscopic peaks, attributable to the major components usually present in ochres, were calculated and compared, one against the another, from the spectra obtained through micro-Raman spectroscopy. Elementary composition is also revealed through a scanning electron microscopy (SEM) analysis. The possibility for detailed study on a particular Byzantine ochre palette can thus be performed based on the small differences in its nature and composition. These differences can first be observed and then measured among all of the natural earth pigments, through microRaman and microFTIR spectroscopies.     

Development of enzyme-linked immunosorbent assays for the detection of deacetoxycephalosporin C and isopenicillin N synthase activity

Although there are a number of existing assays for monitoring the activity of both isopenicillin N synthase (IPNS) and deacetoxycephalosporin C synthase (DAOCS), none have demonstrated the qualities required for screening a mutant library. Hence, enzyme-linked immunosorbent assays (ELISAs) for IPNS and DAOCS were developed based on the detection of the catalytic turnover products isopenicillin N and cephalexin/phenylacetyl-7-aminodeacetoxycephalosporanic acid (G-7-ADCA), respectively. These assays are relatively fast compared to existing assays, such as the hole-plate bioassay, and are amenable with high-throughput screening. Both the IPNS and DAOCS-ELISAs were optimised for use with crude protein extracts rather than purified protein, thereby eliminating any additional time required for purification. The ELISA developed for the detection of cephalexin had an IC₅₀ value of 154 ± 9 ng mL⁻¹ and LOD of 7.2 ± 2.2 ng mL⁻¹ under conditions required for the assay. Good recoveries and correlation was observed for spiked samples when the concentration of crude protein was kept below 1 mg mL⁻¹. The DAOCS-ELISA was found to have increased sensitivity compared to the hole-plate bioassay (10.3 μg mL⁻¹). The IPNS-ELISA did not significantly increase the sensitivity (approximately 5 μg mL⁻¹) compared to that of the hole-plate bioassay (16 μg mL⁻¹) for isopenicillin N. The minimum amount of crude protein extract required for producing detectable amounts of product for both assays was below 0.5% of the maximum amount of protein that the assay could contain without any effect on the ELISA. This suggests that when screening a mutant library, mutants producing low amounts of the product could still be detected using these assays.     

C-24 stereochemistry of marine sterols: (22E)-24-(isopropenyl)-22-dehydrocholesterol and 24-isopropenylcholesterol

The C-24 configuration of (22E,24xi)-24-isopropenyl-22-dehydrocholesterol (1), which was recently isolated from the Colombian Caribbean sponge, Topsentia ophiraphidites, was investigated. Synthesis of the stereodefined (24R)- and (24S)-(22E)-24-isopropenyl-22-dehydrocholesterols (1a, 1b) followed by (1)H- and (13)C-NMR data comparison of these sterols established the (24R)-configuration of 1. In addition, (24R)- and (24S)-24-isopropenylcholesterols (2a and 2b) were also synthesized and their NMR data are provided. The C-24 configurations of the samples of 24-isopropenylcholesterol reported previously are discussed.     

Two Novel Lyso-Ornithine Lipids Isolated from an Arctic Marine Lacinutrix sp. Bacterium

The Lacinutrix genus was discovered in 2005 and includes 12 Gram-negative bacterial species. To the best of our knowledge, the secondary metabolite production potential of this genus has not been explored before, and examination of Lacinutrix species may reveal novel chemistry. As part of a screening project of Arctic marine bacteria, the Lacinutrix sp. strain M09B143 was cultivated, extracted, fractionated and tested for antibacterial and cytotoxic activities. One fraction had antibacterial activity and was subjected to mass spectrometry analysis, which revealed two compounds with elemental composition that did not match any known compounds in databases. This resulted in the identification and isolation of two novel isobranched lyso-ornithine lipids, whose structures were elucidated by mass spectrometry and NMR spectroscopy. Lyso-ornithine lipids consist of a 3-hydroxy fatty acid linked to the alpha amino group of an ornithine amino acid through an amide bond. The fatty acid chains were determined to be iso-C15:0 (1) and iso-C16:0 (2). Compound 1 was active against the Gram-positive S. agalactiae, while 2 showed cytotoxic activity against A2058 human melanoma cells.