Optimization and Verification of Droplet Digital PCR Even-Specific Methods for the Quantification of GM Maize DAS1507 and NK603

In recent years, digital polymerase chain reaction (dPCR), a new molecular biology technique, has been gaining in popularity. Among many other applications, this technique can also be used for the detection and quantification of genetically modified organisms (GMOs) in food and feed. It might replace the currently widely used real-time PCR method (qPCR), by overcoming problems related to the PCR inhibition and the requirement of certified reference materials to be used as a calibrant. In theory, validated qPCR methods can be easily transferred to the dPCR platform. However, optimization of the PCR conditions might be necessary. In this study, we report the transfer of two validated qPCR methods for quantification of maize DAS1507 and NK603 events to the droplet dPCR (ddPCR) platform. After some optimization, both methods have been verified according to the guidance of the European Network of GMO Laboratories (ENGL) on analytical method verification (ENGL working group on “Method Verification.” (2011) Verification of Analytical Methods for GMO Testing When Implementing Interlaboratory Validated Methods). Digital PCR methods performed equally or better than the qPCR methods. Optimized ddPCR methods confirm their suitability for GMO determination in food and feed.     

Optimisation of sample pre-treatment method for the determination of triclosan in marine sediments by high-performance liquid chromatography and marine benthic quality assessment in the southern Baltic Sea

This study comprises optimisation of sample preparation and HPLC analytical procedure for the determination of a personal care product ingredient, triclosan (TCS), in marine sediments. The testing of several varying pre-treatment parameters confirmed that ultrasonic extraction is an effective method for the isolation of TCS from marine sediments, and that the choice of extraction solvent appeared to be of major importance. The selection of the mobile-phase composition and the absorption wavelength was made for the high-performance liquid chromatography analysis step. Based on the validated method, a preliminary assessment of the benthic ecosystem quality with regards to TCS contamination has been demonstrated in the southern Baltic Sea – a semi-enclosed sea, characterised by poor water exchange, thus particularly susceptible to anthropopression. TCS has been identified and quantified in situ in marine bottom sediments, sediment dwelling isopod – Saduria entomon L. and estimated in silico in pore waters based on the equilibrium partition theory in order to assess the potential exposure and uptake from the aqueous phase. TCS concentrations identified in the bottom sediments of the Gdansk Basin, as the natural habitat for studied S.entomon L., appear to be threatening to the benthic environment. Particularly when considering S. entomon L. as a major nutrition source for cod (Gadus morhua) undergoing the feminisation process, since the recent studies prove TCS to have a potential to induce critical alterations in the endocrine system of marine ichthyofauna.     

Drone Brood Homogenate as Natural Remedy for Treating Health Care Problem: A Scientific and Practical Approach

Drone brood homogenate is a little-known bee product used in folk medicine to treat various health problems. It is a very nutritious milky substance with high content of nutrients: proteins, lipids, fatty acids, carbohydrates, vitamins (A, B, E and D), and minerals. Moreover, when collected on early stage of larvae development, it is, most of all, rich source of sex hormone (testosterone, progesterone and estradiol). Some beekeepers consider drone brood as a waste product, although in some countries they use it to fight Varroa. Meanwhile, in many scientific reports a curative effect of bee drone homogenate in treating urgent global health problems have been confirmed, including ovarian dysfunction in women and male infertility, thyroid and immunity disorders, as well as malnutrition in children. A few dietary supplements based on drone brood are available online. Many patents relating to drone brood-based dietary supplements have been filed in Russia, but their prevalence in EU countries is still limited. Further research is needed to fully recognize the pharmacological activity and increase the use of drone brood.     

Labeled vs. Label-Free Raman Imaging of Lipids in Endothelial Cells of Various Origins

Endothelial cells (EC) constitute a single layer of the lining of blood vessels and play an important role in maintaining cardiovascular homeostasis. Endothelial dysfunction has been recognized as a primary or secondary cause of many diseases and it manifests itself, among others, by increased lipid content or a change in the lipid composition in the EC. Therefore, the analysis of cellular lipids is crucial to understand the mechanisms of disease development. Tumor necrosis factor alpha (TNF-α)-induced inflammation of EC alters the lipid content of cells, which can be detected by Raman spectroscopy. By default, lipid detection is carried out in a label-free manner, and these compounds are recognized based on their spectral profile characteristics. We consider (3S,3′S)-astaxanthin (AXT), a natural dye with a characteristic resonance spectrum, as a new Raman probe for the detection of lipids in the EC of various vascular beds, i.e., the aorta, brain and heart. AXT colocalizes with lipids in cells, enabling imaging of lipid-rich cellular components in a time-dependent manner using laser power 10 times lower than that commonly used to measure biological samples. The results show that AXT can be used to study lipids distribution in EC at various locations, suggesting its use as a universal probe for studying cellular lipids using Raman spectroscopy. The use of labeled Raman imaging of lipids in the EC of various organs could contribute to their easier identification and to a better understanding of the development and progression of various vascular diseases, and it could also potentially improve their diagnosis and treatment.     

Development and validation method for the determination of selected tetracyclines in animal medicated feedingstuffs with the use of micellar liquid chromatography

A chromatographic procedure for the determination of oxytetracycline (OTC), tetracycline (TC), chlorotetracycline (CTC), and doxycycline (DC) in medicated feedingstuffs was developed. Samples were extracted with 0.01 M citric buffer/acetonitrile (pH 3.0) and further purified with 0.45 μm syringe filters. The purified extract was separated on Thermo column C₁₈, 150?×?4 mm, 5 μm and detection was carried out at 360 nm for OTC, and TC, 370 nm for CTC, and 350 nm for DC. TCs were eluted with a mobile phase of 0.03 M SDS/7 % 1-butanol/0.02 M oxalic acid/NaOH at pH 2.5. This method provided average recoveries of 80.4 % to 100.2 %, with CVs of 0.5 % to 6.6 % in the range of 50 to 1500 mg/kg OTC, TC, CTC, and DC in feeds. The linearity for the four TCs was determined by high-performance liquid chromatography-diode array detector (HPLC-DAD) in the range 10–300 μg/mL (50–1500 mg/kg), with a linear correlation coefficient (R)?>?0.99. The LOD and LOQ for TCs in pig and poultry feeds ranged from 4.0 to 10.7 and 4.7 to 12.6 mg/kg, respectively. The methodology was applied to the analysis of animal feedingstuffs collected from poultry and pig farms.

Application of Micellar Mobile Phase for Quantification of Sulfonamides in Medicated Feeds by HPLC-DAD

Rapid chromatographic procedure for quantification of five sulfonamides in medicated feeds are proposed. Satisfactory separation of sulfonamides from medicated feeds was achieved using a Zorbax Eclipse XDB C18 column (4.6 × 150 mm, 5 µm particle size) with a micellar mobile phase consisting of 0.05 M sodium dodecyl sulphate, 0.02 M phosphate buffer, and 6% propan-2-ol (pH 3). UV quantitation was set at 260 nm. The proposed procedure allows the determination of sulfaguanidine, sulfadiazine, sulfamerazine, sulfamethazine, and sulfamethoxazole in medicated feeds for pigs and poultry. Application of the proposed method to the analysis of five pharmaceuticals gave recoveries between 72.7% to 94.7% and coefficients of variations for repeatability and reproducibility between 2.9% to 9.8% respectively, in the range of 200 to 2000 mg/kg sulfonamides in feeds. Limit of detection and limit of quantification were 32.7–56.3 and 54.8–98.4 mg/kg, respectively, depending on the analyte. The proposed procedure for the quantification of sulfonamides is simple, rapid, sensitive, free from interferences and suitable for the routine control of feeds. In the world literature, we did not find the described method of quantitative determination of sulfonamides in medicated feeds with the use of micellar liquid chromatography.     

XMCD studies of magnetic polarization at Mo atoms in CoMo alloy and magnetically coupled Co/Mo multilayers

Magnetic polarization of Mo atoms in Co₉₆Mo₄ alloy film and Co/Mo multilayered structures has been studied by X‐ray magnetic circular dichroism. Samples with Mo spacers of two different thicknesses (0.9 nm and 1.8 nm) were investigated. Mo atoms receive a magnetic moment of ?0.21μ_B in the alloy. In the multilayer with the thinner Mo spacer (d_Mo = 0.9 nm) the magnetic moment is much smaller (?0.03μ_B). In both cases the measured induced moment at the Mo site is oriented antiparallel to the moment at the Co atoms. The presence of the induced moment in the Mo spacer coincides with antiferromagnetic coupling between the Co component slabs. In contrast, neither measurable induced moment at the Mo site nor interlayer coupling between the Co layers has been found for the multilayer with the thicker Mo spacer. Possible mechanisms of the coupling associated with the induced moment are discussed in detail.     

Kinetics of refractive index changes in polymeric photochromic films

Methacrylate copolymers containing, in side chain azobenzene groupings with heterocyclic sulfonamide substituents: N-(2,6-dimethylpyrimidin-4-yl)sulfamoyl (sulfisomidine) and N-(5-methylisoxazol-3-yl) sulfamoyl (sulfamethoxazole) were investigated. The materials undergo reversible trans-cis isomerisation during illumination with light. This results in changes of dipole moment, polarizability and refractive index. Ellipsometric measurements showed a distinct decreasing refractive index during illumination with light corresponding to absorption band (ca. 450 nm). Depending on the polymer, the change of real part of refractive index in spin-coated films was between 0.016 and 0.031. The dynamics of growth and decay of refractive index changes, was described by biexponential function approach.     

Bispalladium(II) Complexes of di-p-Pyrirubyrin Derivatives as Promising Near-Infrared Photoacoustic Dyes

The insertion of palladium(II) into di-p-pyrirubyrin results in mutually convertible bimetallic complexes. Post-synthetic functionalization of one of them yielded bispalladium(II) dioxo-di-p-pyrirubyrin and, after demetallation, dioxo-di-p-pyrirubyrin, introducing for the first time the α,β′-pyridin-2-one unit into the macrocyclic frame. Bispalladium(II) di-p-pyrirubyrin 6 , bispalladium(II) dioxo-di-p-pyrirubyrin 9 , and dioxo-di-p-pyrirubyrin 10 absorb and emit light around 1000 nm and are characterized by high photostability. Thus, they are promising candidates for near-infrared photoacoustic dyes, ideally targeting ( 9 ) the wavelength of Yb-based fiber lasers. The incorporation of an α,β′-pyridine moiety into expanded porphyrins opens a highly interesting area of research due to the attractive optical and coordination properties of the resulting molecules.