Structure of the nitrogen-centered radical formed during inactivation of E. coli ribonucleotide reductase by 2'-azido-2'-deoxyuridine-5'-diphosphate: trapping of the 3'-ketonucleotide

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides providing the monomeric precursors required for DNA replication and repair. The class I RNRs are composed of two homodimeric subunits: R1 and R2. R1 has the active site where nucleotide reduction occurs, and R2 contains the diiron tyrosyl radical (Y*) cofactor essential for radical initiation on R1. Mechanism-based inhibitors, such as 2'-azido-2'-deoxyuridine-5'-diphosphate (N(3)UDP), have provided much insight into the reduction mechanism. N(3)UDP is a stoichiometric inactivator that, upon interaction with RNR, results in loss of the Y* in R2 and formation of a nitrogen-centered radical (N*) covalently attached to C225 (R-S-N*-X) in the active site of R1. N(2) is lost prior to N* formation, and after its formation, stoichiometric amounts of 2-methylene-3-furanone, pyrophosphate, and uracil are also generated. On the basis of the hyperfine interactions associated with N*, it was proposed that N* is also covalently attached to the nucleotide through either the oxygen of the 3'-OH (R-S-N*-O-R') or the 3'-C (R-S-N*-C-OH). To distinguish between the proposed structures, the inactivation was carried out with 3'-[(17)O]-N(3)UDP and N* was examined by 9 and 140 GHz EPR spectroscopy. Broadening of the N* signal was detected and the spectrum simulated to obtain the [(17)O] hyperfine tensor. DFT calculations were employed to determine which structures are in best agreement with the simulated hyperfine tensor and our previous ESEEM data. The results are most consistent with the R-S-N*-C-OH structure and provide evidence for the trapping of a 3'-ketonucleotide in the reduction process.     

Review of the Sampling and Pretreatment Methods for Dioxins Determination in Solids,Liquids and Gases

The analytical requirements for the determination of dioxins (e.g. PCDD, PCDF) are unique in comparison with those for other routinely monitored chemicals: dioxins are measured in parts per trillion (ppt) and lower levels in technical and environmental samples, whereas other organics are measured in parts per million (ppm) or parts per billion (ppb). The ultra‐trace analysis of dioxins requires a high sample enrichment obtainable by the use of efficient extraction methods and highly selective purification/fractionation techniques. Furthermore, highly sensitive equipment (e.g. high resolution gas chromatography ‐ HRGC) with detection levels in the ppt range must be used to isolate and separate a huge number of dioxin congeners. The quantification of the dioxins by a high resolution mass detector (HRMS) requires the use of internal and labeled standards. This paper reviews and compares the classic and modern pretreatment methods, namely extraction and clean‐up, that are currently used in dioxin analysis in industrial samples and that were improved or developed in the last 25 years. In addition, the standard methods for dioxin sampling from an emission source (flue or stack gas) are examined and compared with new, automated techniques.     

Attomole-per Cell Atomic Mass Spectrometry Measurement of Platinum and Gold Drugs in Cultured Lung Cancer Cells

In this study, we developed a strategy to determine atto- and femtomolar amounts of metal ions in lysates and mineralizates of cells (human non-small-cell lung carcinoma (NSCLC, A549) and normal lung (MRC-5)) exposed to cytotoxic metallo-drugs: cisplatin and auranofin at concentrations close to the half-maximal inhibitory drug concentrations (IC₅₀). The developed strategy combines data obtained using biological and chemical approaches. Cell density was determined using two independent cell staining assays using trypan blue, calcein AM/propidium iodide. Metal concentrations in lysed and mineralized cells were established employing a mass spectrometer with inductively coupled plasma (ICP-MS) and equipped with a cross-flow nebulizer working in aspiration mode. It allowed for detecting of less than 1 fg of metal per cell. To decrease the required amount of sample material (from 1.5 mL to ~100 µL) without loss of sensitivity, the sample was introduced as a narrow band into a constant stream of liquid (flow-injection analysis). It was noticed that the selectivity of cisplatin accumulation by cells depends on the incubation time. This complex is accumulated by cells at a lower efficiency than auranofin and is found primarily in the lysate representing the cytosol. In contrast, auranofin interacts with water-insoluble compounds. Despite their different mechanism of action, both metallo-drugs increased the accumulation of transition metal ions responsible for oxidative stress.     

Physicochemical Properties,Fatty Acid Composition,Volatile Compounds of Blueberries,Cranberries, Raspberries,and Cuckooflower Seeds Obtained Using Sonication Method

Every year, thousands of tons of fruit seeds are discarded as agro-industrial by-products around the world. Fruit seeds are an excellent source of oils, monounsaturated fatty acids, and n-6 and n-3 polyunsaturated essential fatty acids. This study aimed to develop a novel technology for extracting active substances from selected seeds that were obtained after pressing fruit juices. The proposed technology involved sonification with the use of ethyl alcohol at a low extraction temperature. Seeds of four species—blueberry (Vaccinium myrtillus L.), raspberry (Rubus idaeus), cranberry (Vaccinium macrocarpon), and cuckooflower (Cardamine pratensis)—were used for extraction. Following alcohol evaporation under nitrogen, the antioxidant activity, chemical composition, and volatile compounds of the obtained extracts were analyzed using chromatographic methods, including gas chromatography (GC)–mass spectrometry (MS) (GC–MS/MS), and high-performance liquid chromatography–MS. We analyzed physicochemical properties, fatty acid, and volatile compounds composition, sterol and tocochromanol content of blueberry, cranberry, raspberry, and cuckooflower seed oils obtained by sonication. This method is safe and effective, and allows for obtaining valuable oils from the seeds.     

Chemical Characteristics and Antioxidant Activity of Arctostaphylos uva-ursi L. Spreng. at the Southern Border of the Geographical Range of the Species in Europe

The bearberry (Arctostaphylos uva-ursi L. Spreng.) is a source of herbal material—bearberry leaf (Uvae ursi folium), which is highly valued and sought by pharmaceutical and cosmetic industries. For many years, leaves of this plant have been used in traditional medicine as a diuretic, antimicrobial, and anti-inflammatory agent for various diseases of the urogenital tract. The bearberry has also been proposed as a natural antioxidant additive due to the high contents of phenolic compounds in its leaves. The study was focused on characterization of the basic phytochemical composition and antioxidant activity of extracts derived from bearberry leaves collected from plants located at the southern border of the geographical range of the species in Europe. The investigated herbal material is characterized by a different chemical profile compared to the chemical profiles of bearberry found in other parts of the continent. Bearberry extracts from plants growing in two different habitat types—heathlands and pine forests showed a wide range of variation, especially in the concentration of hyperoside, corilagin, and methylartutin and the total flavonoid contents. In addition to arbutin, bearberry can be a valuable source of phenolic compounds, which are mainly responsible for the antioxidant properties of extracts. The high content of phenols and high values of antioxidant parameters indicate a high potential of bearberry leaves to be used as a powerful natural source of antioxidants in herbal preparations. Therefore, the A. uva-ursi populations can be a source of plant material for pharmaceutical, cosmetic, and food industries.     

Investigation of nonlinear absorption in YAP and YAG single crystals doped with Co and co-doped with Si

Since our attempts to grow YAP:Co,Si non-linear absorbers have failed, we switched to Czochralski grown YAGs doped with the same ions. This time, however, the new material was found to exhibit excellent, non-linear absorption, which can be used directly in Q-switching elements in laser systems. In this work we report on technology of YAG single crystals doped with Co and Si, their optical properties, and, finally, on their non-linear absorption. An explanation on why the YAPs were found to be inefficient in non-linear absorption was also provided.     

Tb3Sc2Al3O12-TbScO3 eutectic self-organized microstructure for metamaterials and photonic crystals application

Eutectics are the materials with foreseen application in the field of photonic crystals and metamaterials. In this paper, the dependence on chemical composition of the microstructures of terbium-scandium-aluminium gamet and terbium-scandium perovskite (Tb₃Sc₂Al₃O₁₂-TbScO₃) eutectics has been studied. The growth of the eutectic rods by the micro-pulling down method is presented, using compositions with several different volume fractions of the garnet and the perovskite phases, V_TSAG:V_TSP = 4, 3, 2, 1, 1/2. The phases have been characterized by powder X-ray diffraction and energy dispersive spectrometry. The relationship between the lattice constant of individual phases and the chemical composition is presented. The unidirectional growth of microrods has been also investigated by electron backscattering diffraction.     

Correlations of neutral and charged particles in 40Ar- 58Ni reaction at 77 MeV/u

The measurement of the two-particle correlation function for different particle species allows to obtain information about the development of the particle emission process: the space-time properties of emitting sources and the emission time sequence of different particles. The single-particle characteristics and two-particle correlation functions for neutral and charged particles registered in forward direction are used to determine that the heavy fragments (deuterons and tritons) are emitted in the first stage of the reaction (pre-equilibrium source) while the majority of neutrons and protons originates from the long-lived quasi-projectile. The emission time sequence of protons, neutrons and deuterons has been obtained from the analysis of non-identical particle correlation functions.     

Whole genome amplification induced bias in the detection of KRAS‐mutated cell populations during colorectal carcinoma tissue testing

Whole genome amplification replicates the entire DNA content of a sample and can thus help to circumvent material limitations when insufficient DNA is available for planned genetic analyses. However, there are conflicting data in the literature whether whole genome amplification introduces bias or reflects precisely the spectrum of starting DNA. We analyzed the origins of discrepancies in KRAS (Kirsten rat sarcoma viral oncogene homolog gene) mutation detection in six of ten samples amplified using the GenomePlex® Tissue Whole Genome Amplification kit 5 (WGA5; Sigma‐Aldrich, St. Louis, MO, USA) and KRAS StripAssay® (KRAS SA; ViennaLab Diagnostics, Vienna, Austria). We undertook reextraction, reamplification, retyping, authentication, reanalysis, and reinterpretation to determine whether the discrepancies originated during the preanalytical, analytical, and/or interpretative phase of genotyping. We conclude that a combination of glass slide/sample heterogeneity and biased amplification due to stochastic effects in the early phases of whole genome amplification (WGA) may have adversely affected the results obtained. Our findings are relevant for both forensic genetics testing and massively parallel sequencing using preamplification.