Mutation scanning-based analysis of anisakid larvae from Sillago flindersi from Bass Strait, Australia

Anisakidosis is an important fish-borne disease caused by the larvae of anisakid nematodes, which affects humans and a range of other animals. The accurate identification of members of this nematode group is central to investigating the epidemiology of the parasites and in the surveillance and control of anisakidosis. It is now well known that morphological identification alone does not allow specific identification, particularly of larval stages. To better understand the epidemiology of anisakid nematodes in southern Australian fishes and the potential risks posed to human health, a survey of 50 specimens of the commercially important fish, Sillago flindersi, from Bass Strait, Australia was conducted. We characterised anisakid larvae by PCR-coupled mutation scanning, sequencing and phylogenetic analyses of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA. This study revealed that 92% of the S. flindersi examined were infected with anisakids (n=194), which were represented by seven genotypes. Phylogenetic analyses of the genotypes defined herein, together with reference sequence for Anisakis pegreffii and Hysterothylacium sp. from public databases (i.e. GenBank), revealed the presence of A. pegreffii (n=24), Hysterothylacium larval type IV (n=90) and Hysterothylacium larval type VIII (n=80) in S. flindersi. Thus, the PCR-coupled mutation scanning approach employed herein is an effective tool for the genetic characterisation of anisakid nematodes for diagnostic and analytical purposes (nucleotide sequences reported in this paper are available in the GenBank database under accession nos. JN631796-809).     

Molecular dynamics simulations reveal specific interactions of post-translational palmitoyl modifications with rhodopsin in membranes

We present a detailed analysis of the behavior of the highly flexible post-translational lipid modifications of rhodopsin from multiple-microsecond all-atom molecular dynamics simulations. Rhodopsin was studied in a realistic membrane environment that includes cholesterol, as well as saturated and polyunsaturated lipids with phosphocholine and phosphoethanolamine headgroups. The simulation reveals striking differences between the palmitoylations at Cys322 and Cys323 as well as between the palmitoyl chains and the neighboring lipids. Notably the palmitoyl group at Cys322 shows considerably greater contact with helix H1 of rhodopsin, yielding frequent chain upturns with longer reorientational correlation times, and relatively low order parameters. While the palmitoylation at Cys323 makes fewer protein contacts and has increased order compared to Cys322, it nevertheless exhibits greater flexibility with smaller order parameters than the stearoyl chains of the surrounding lipids. The dynamical structure of the palmitoylations-as well as their extensive fluctuations-suggests a complex function for the post-translational modifications in rhodopsin and potentially other G protein-coupled receptors, going beyond their role as membrane anchoring elements. Rather, we propose that the palmitoylation at Cys323 has a potential role as a lipid anchor, whereas the palmitoyl-protein interaction observed for Cys322 suggests a more specific interaction that affects the stability of the dark state of rhodopsin.     

Electrochemically induced oxygen spillover and diffusion on Pt(111): PEEM imaging and kinetic modelling

Electrochemically induced oxygen spillover and diffusion in the Pt(O(2))|YSZ system is investigated in a combined experimental and theoretical study. The spreading of spillover oxygen is imaged by photoelectron emission microscopy (PEEM) on dense and epitaxial Pt(111) thin film electrodes prepared by pulsed laser deposition (PLD). Two different models are used to obtain surface diffusion coefficients from the experimental data, (i) an analytical solution of Fick's 2nd law of diffusion, and (ii) a numerical reaction-diffusion model that includes recombinative desorption of O(2) into the gas phase. The resulting diffusion coefficient has an activation energy of 50 kJ mol(-1) and a preexponential factor of 0.129 cm(2) s(-1) with an estimated uncertainty of ±20% for the activation energy and ±50% for the absolute value. The Fickian model slightly overpredicts diffusion coefficients due to the neglect of oxygen desorption. Experimental and theoretical results and limitations are discussed and compared to previous work.     

Recrystallization of Cu-poor CuInS2 assisted by metallic Cu or Ag

We monitor the recrystallization of Cu-poor CuInS₂ thin films assisted by pure Cu or pure Ag by means of real-time synchrotron-based polychromatic X-ray diffraction. In both cases a new microstructure is formed accompanied by an increase in grain size. In the case of Cu, the onset temperature of the thin-film recrystallization is higher than 370 °C. In the case of Ag, the thin-film recrystallization comes to an end at 270 °C. The Ag-assisted recrystallization occurs in the presence of the body-centered cubic β-Ag₂S phase. We find that domain growth and diffusion of silver into the film occur simultaneously.     

Direct functionalization of indoles: copper-catalyzed malonyl carbenoid insertions

Indoles, when treated with dimethyl diazomalonate under catalysis by Cu(acac)(2), undergo C-H insertion reactions regioselectively depending on the substitution pattern on the indole moiety. Indoles where the 3-position is substituted give high yields of the C2-H insertion product. Microwave conditions are also disclosed which show comparable yields with reduced reaction times.     

Conducting properties of In2O3:Sn thin films at low temperatures

Electrical conductivity, Hall effect and magnetoresistance of In₂O₃:Sn thin films deposited on a glass substrates at different temperatures and oxygen pressures, have been investigated in the temperature range 4.2–300 K. The observed temperature dependences of resistivity for films deposited at 230 °C as well as at nominally room temperatures were typical for metallic transport of electrons except temperature dependence of resistivity of the In₂O₃:Sn film deposited in the oxygen deficient atmosphere. The electrical measurements were accompanied by AFM and SEM studies of structural properties, as well as by XPS analysis. It is established that changes of morphology and crystallinity of ITO films modify the low-temperature behavior of resistivity, which still remains typical for metallic transport. This is not the case for the oxygen deficient ITO layer. XPS analysis shows that grown in situ oxygen deficient ITO films have enhanced DOS between the Fermi level and the valence band edge. The extra localized states behave as acceptors leading to a compensation of n-type ITO. That can explain lower n-type conductivity in this material crossing over to a Mott-type hopping at low temperatures. Results for the low temperature measurements of stoichiometric ITO layers indicate that they do not show any trace of metal-to-insulator transition even at 4.2 K. We conclude that, although ITO is considered as a highly doped wide-band gap semiconductor, its low-temperature properties are very different from those of conventional highly doped semiconductors.     

Screening for illicit and medicinal drugs in whole blood using fully automated SPE and ultra‐high‐performance liquid chromatography with TOF‐MS with data‐independent acquisition

A broad forensic screening method for 256 analytes in whole blood based on a fully automated SPE robotic extraction and ultra‐high‐performance liquid chromatography (UHPLC) with TOF‐MS with data‐independent acquisition has been developed. The limit of identification was evaluated for all 256 compounds and 95 of these compounds were validated with regard to matrix effects, extraction recovery, and process efficiency. The limit of identification ranged from 0.001 to 0.1 mg/kg, and the process efficiency exceeded 50% for 73 of the 95 analytes. As an example of application, 1335 forensic traffic cases were analyzed with the presented screening method. Of these, 992 cases (74%) were positive for one or more traffic‐relevant drugs above the Danish legal limits. Commonly abused drugs such as amphetamine, cocaine, and frequent types of benzodiazepines were the major findings. Nineteen less frequently encountered drugs were detected e.g. buprenorphine, butylone, cathine, fentanyl, lysergic acid diethylamide, m‐chlorophenylpiperazine, 3,4‐methylenedioxypyrovalerone, mephedrone, 4‐methylamphetamine, p‐fluoroamphetamine, and p‐methoxy‐N‐methylamphetamine. In conclusion, using UHPLC–TOF‐MS screening with data‐independent acquisition resulted in the detection of common drugs of abuse as well as new designer drugs and more rarely occurring drugs. Thus, TOF‐MS screening of blood samples constitutes a practical way for screening traffic cases, with the exception of δ‐9‐tetrahydrocannabinol, which should be handled in a separate method.     

A fast and inexpensive procedure for the isolation of synthetic cannabinoids from ‘Spice’ products using a flash chromatography system

In the age of the Internet, the variety of drugs offered online is constantly increasing, and new drugs emerge every month. One group of drugs showing such an enormous increase is that of synthetic cannabinoids. Since their first identification in ‘herbal mixtures’, new structural modifications continue to appear on the market. In order to keep up with this process, toxicological screening methods need to be up to date. This can become extremely difficult if no reference material is available. In this article, a fast and effective way to extract and purify synthetic cannabinoids from ‘herbal mixtures’ is presented. This method opens a new opportunity for a timely reaction by obtaining reference material straight out of the ‘herbal mixtures’ ordered via the Internet. Isolation was carried out on a flash chromatography system with gradient elution on a C18 column using methanol and 0.55 % formic acid as mobile phases. The obtained purity of all compounds exceeded 99 %. In addition to the isolation of single compounds, the method proved to be suitable for the separation of various synthetic cannabinoids in one mixture, including the diastereomers cis- and trans-CP-47,497-C8. This approach for obtaining pure standards of new drugs proved to be effective, inexpensive and much quicker than waiting for the substances to be commercially available as reference material.

Continuous Selection and Unique Polyhedral Representation of Solutions to Convex Parametric Quadratic Programs

A method for obtaining continuous solutions to convex quadratic and linear programs with parameters in the linear part of the objective function and right-hand side of the constraints is presented. For parameter values for which the problem has nonunique solutions, the optimizer with the least Euclidean norm is selected. The normal cone optimality condition is utilized to obtain a unique polyhedral representation of the piecewise affine minimizer function. This research is part of the Strategic University Program on Computational Methods for Nonlinear Motion Control funded by the Research Council of Norway. We thank Dr. E.C. Kerrigan at the Department of Electrical Engineering, Imperial College, London and Dr. Colin Jones at ETH Zürich for their comments. Finally, we thank the anonymous reviewers for their comments.