Comparison of the in vitro assays to investigate the hepatic metabolism of seven pesticides in Cyprinus carpio and Oncorhynchus mykiss.

Liver S9 fractions from common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) were incubated with seven pesticides (fenamidone, fenoxaprop-p-ethyl, penflufen, spirotetramat, tebuconazole, tembotrione and trifloxystrobin) and the metabolic pathways of the applied chemicals were determined by HPLC-high-resolution mass spectrometry. Five of the seven pesticides (fenamidone, penflufen, spirotetramat, trifloxystrobin and fenoxaprop-p-ethyl) revealed a higher metabolic capacity of rainbow trout liver fractions compared to carp liver fractions. The other two pesticides (tebuconazole and tembotrione) showed a similar and marginal biotransformation for liver S9 fractions of both species.

A toxicogenomics approach reveals characteristics supporting the honey bee (Apis mellifera L.) safety profile of the butenolide insecticide flupyradifurone.

Flupyradifurone, a novel butenolide insecticide, selectively targets insect nicotinic acetylcholine receptors (nAChRs), comparable to structurally different insecticidal chemotypes such as neonicotinoids and sulfoximines. However, flupyradifurone was shown in acute toxicity tests to be several orders of magnitude less toxic to western honey bee (Apis mellifera L.) than many other insecticides targeting insect nAChRs.

Identification and functional characterisation of a novel N-cyanoamidine neonicotinoid metabolising cytochrome P450, CYP9Q6, from the buff-tailed bumblebee Bombus terrestris.

Recent work has shown that two bumblebee (Bombus terrestris) cytochrome P450s of the CYP9Q subfamily, CYP9Q4 and CYP9Q5, are important biochemical determinants of sensitivity to neonicotinoid insecticides. Here, we report the characterisation of a third P450 gene CYP9Q6, previously mis-annotated in the genome of B. terrestris, encoding an enzyme that metabolises the N-cyanoamidine neonicotinoids thiacloprid and acetamiprid with high efficiency.

Genomic insights into neonicotinoid sensitivity in the solitary bee Osmia bicornis.

The impact of pesticides on the health of bee pollinators is determined in part by the capacity of bee detoxification systems to convert these compounds to less toxic forms. For example, recent work has shown that cytochrome P450s of the CYP9Q subfamily are critically important in defining the sensitivity of honey bees and bumblebees to pesticides, including neonicotinoid insecticides. However, it is currently unclear if solitary bees have functional equivalents of these enzymes with potentially serious implications in relation to their capacity to metabolise certain insecticides.

Unravelling the Molecular Determinants of Bee Sensitivity to Neonicotinoid Insecticides.

The impact of neonicotinoid insecticides on the health of bee pollinators is a topic of intensive research and considerable current debate [1]. As insecticides, certain neonicotinoids, i.e.

Proteomic profiling of lung immune cells reveals dysregulation of phagocytotic pathways in female-dominated molecular COPD phenotype.

Background: Smoking is the main risk factor for chronic obstructive pulmonary disease (COPD). Women with COPD who smoke experienced a higher risk of hospitalization and worse decline of lung function. Yet the mechanisms of these gender-related differences in clinical presentations in COPD remain unknown.

Neofunctionalization of Duplicated P450 Genes Drives the Evolution of Insecticide Resistance in the Brown Planthopper.

Gene duplication is a major source of genetic variation that has been shown to underpin the evolution of a wide range of adaptive traits [1, 2]. For example, duplication or amplification of genes encoding detoxification enzymes has been shown to play an important role in the evolution of insecticide resistance [3-5]. In this context, gene duplication performs an adaptive function as a result of its effects on gene dosage and not as a source of functional novelty [3, 6-8].

Approach for Identifying Human Leukocyte Antigen (HLA)-DR Bound Peptides from Scarce Clinical Samples.

Immune-mediated diseases strongly associating with human leukocyte antigen (HLA) alleles are likely linked to specific antigens. These antigens are presented to T cells in the form of peptides bound to HLA molecules on antigen presenting cells, e.g.

Effects of exercise on the urinary proteome.

Exercise-induced proteinuria has been observed and studied for more than a century. It was found that different sport disciplines alter the urinary proteome in different ways. Moderate-intensity exercise results in increased glomerular filtration, meaning that medium-sized proteins are excreted in higher amounts, while high-intensity exercise of short duration also increases the excretion of low molecular weight proteins as a result of tubular dysfunction.

Analysis of intraspecific variation in venoms of Acanthophis antarcticus death adders from South Australia.

Intraspecific variation in venom composition and activity has been reported from a wide range of snakes. Geographical origin can be one cause for this variation and has recently been documented from Acanthophis antarcticus death adders sampled across four different Australian states. The present study examined whether a narrower sampling range of A.

Detection of small interfering RNA (siRNA) by mass spectrometry procedures in doping controls.

Uncovering manipulation of athletic performance via small interfering (si)RNA is an emerging field in sports drug testing. Due to the potential to principally knock down every target gene in the organism by means of the RNA interference pathway, this facet of gene doping has become a realistic scenario. In the present study, two distinct model siRNAs comprising 21 nucleotides were designed as double strands which were perfect counterparts to a sequence of the respective messenger RNA coding the muscle regulator myostatin of Rattus norvegicus.

Effects of gamma irradiation and 15 days of subsequent ex vivo storage on the cytosolic red blood cell proteome analyzed by 2D-DIGE and Orbitrap MS.

Purpose: Gamma irradiation of red blood cell (RBC) concentrates is routinely used to prevent transfusion-associated graft-versus-host disease. So far, the effects of ionizing radiation on RBC structure and function and especially the proteome are not fully understood.

Experimental Design: RBC concentrates were irradiated with 30 Gy and stored for 1 or 15 days at 4 ± 2°C.

Gender differences in the bronchoalveolar lavage cell proteome of patients with chronic obstructive pulmonary disease.

Background: Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide and is increasing, primarily among women. Underdiagnosis is common, and because of the heterogeneous disease characteristics, molecular markers of specific disease phenotypes and more efficacious treatment regimens are urgently needed.

Objective: In this study the soluble proteome of bronchoalveolar lavage cells, primarily consisting of macrophages, was investigated with the aim of identifying phenotypic differences in early disease development.

Storage-induced changes of the cytosolic red blood cell proteome analyzed by 2D DIGE and high-resolution/high-accuracy MS.

The storage of packed red blood cells (RBCs) is associated with the development of morphological and biochemical changes leading to a reduced posttransfusion functionality and viability of the cells. Within this study, 2D DIGE and high-resolution/high-accuracy Orbitrap MS were used to analyze the storage-induced changes of the cytosolic RBC proteome and identify characteristic protein patterns and potential marker proteins for the assessment of RBC storage lesions. Leukodepleted RBC concentrates were stored according to standard blood bank conditions for 0, 7, 14, 28, and 42 days and analyzed by using a characterized and validated protocol.

Validated hemoglobin-depletion approach for red blood cell lysate proteome analysis by means of 2D PAGE and Orbitrap MS.

The analysis of the cytosolic red blood cell (RBC) proteome is negatively affected by the high intracellular amount of hemoglobin complicating the detection of low-abundant cytosolic proteins. In this study, an alternative approach for the preparation of hemoglobin-depleted RBC lysates is presented, which was established in combination with downstream 2D PAGE analysis and Orbitrap MS. Hemoglobin removal was accomplished by using HemoVoid(TM) depletion reagent, which enabled a very efficient enrichment of low-abundant proteins by simultaneously reducing the hemoglobin concentration of the sample.

RNA interference for performance enhancement and detection in doping control.

RNA interference represents a comparably new route of regulating and manipulating specific gene expression. Promising results were obtained in experimental therapies aim at the treatment of different kinds of diseases including cancer, diabetes mellitus or Dychenne muscular dystrophy. While studies on down-regulation efficiency are often performed by analyzing the regulated protein, the direct detection of small, interfering RNA molecules and antisense oligonucleotides is of great interest for the investigation of the metabolism and degradation and also for the detection of a putative misuse of these molecules in sports.

Confiscated black market products and nutritional supplements with non-approved ingredients analyzed in the Cologne Doping Control Laboratory 2009.

Doping control laboratories are frequently confronted with new substances that may be misused by athletes. Besides new pharmaceuticals, where method development for their detection is dependent on the availability of the substance and corresponding administration studies, some professional and amateur athletes are using illicit 'black market' products, which either differ from known pharmaceuticals but cause similar effects or still are undergoing clinical trials and are therefore rarely available to doping control laboratories. In the Cologne Doping Control Laboratory, different confiscated products and legally obtained nutritional supplements were analyzed in 2009, and various findings were reported including GH-labelled injection vials without any pharmacologically active content; combinations of products indicating the attempt to mask growth hormone abuse; unpurified long-R(3) -IGF-1; nutritional supplements containing the growth hormone releasing peptide-2 (GHRP-2); and ampoules containing the selective androgen receptor modulator Andarine (S-4).

Determination of IGF-1 and IGF-2, their degradation products and synthetic analogues in urine by LC-MS/MS.

Peptide analysis in doping controls by means of nano-UPLC coupled high resolution/high mass accuracy mass spectrometry provides the state-of-the-art technique in modern sports drug testing. The present study describes a recent application of this technique for the qualitative determination of different urinary insulin-like growth factor (IGF) related peptides. After simultaneous isolation by solid phase extraction and magnetic particle-based immunoaffinity purification, target analytes (IGF-1, IGF-2, Des1-3-IGF-1, R(3)-IGF-1 and longR(3)-IGF-1) were separated by nano-liquid chromatography prior to mass spectrometric detection.

Effects of endurance exercise on the urinary proteome analyzed by 2-D PAGE and Orbitrap MS.

Purpose: Exercise-induced proteinuria is a well-known phenomenon and the influence of parameters such as intensity and duration was studied extensively. Usually, total protein or albumin was measured for diagnosis of a proteinuria, and the present study was performed to search for qualitative differences in the urinary proteome before and after endurance exercise.

Experimental Design: Urine samples were concentrated and proteins separated by means of 2-D PAGE.

Identification of the growth-hormone-releasing peptide-2 (GHRP-2) in a nutritional supplement.

Black market products of a pharmaceutical nature and nutritional supplements have received substantial and increasing attention because of potential performance enhancement in elite and non-professional sports. In addition, improved general health is claimed for non-competing individuals. The risks and foreseeable dangers of the uncontrolled use of highly potent and non-approved pharmaceutical compounds in healthy individuals are of considerable concern.

Detection of His-tagged Long-R³-IGF-I in a black market product.

Objective: Performance-enhancing substances are illicitly used in elite or amateur sports and may be obtained from the black market due to a cheaper and easier availability. Although various studies have shown that black market products frequently do not contain the declared substances, enormous amounts of illegally produced and/or imported drugs are confiscated from athletes or at customs with alarming results concerning the outcome of the analyses of the ingredients. This case report describes the identification of His-tagged Long-R³-IGF-I, which is usually produced for biochemical studies, in an injection vial.

Mass spectrometric detection of siRNA in plasma samples for doping control purposes.

Small interfering ribonucleic acid (siRNA) molecules can effect the expression of any gene by inducing the degradation of mRNA. Therefore, these molecules can be of interest for illicit performance enhancement in sports by affecting different metabolic pathways. An example of an efficient performance-enhancing gene knockdown is the myostatin gene that regulates muscle growth.

Mass spectrometry-based characterization of new drugs and methods of performance manipulation in doping control analysis.

Efficient and comprehensive sports drug testing necessitates frequent updating and proactive, preventive anti-doping research, and the early implementation of new, emerging drugs into routine doping controls is an essential aspect. Several new drugs and drug candidates with potential for abuse, including so-called Rycals (ryanodine receptor calstabin complex stabilizers, for example, S-107), hypoxia-inducible factor (HIF) stabilizers, and peroxisome-proliferator-activated receptor (PPAR) delta agonists (for example, GW1516), were studied using different mass spectrometry- and ion mobility-based approaches, and their gas phase dissociation behaviors were elucidated. The detailed knowledge of fragmentation routes allows a more rapid identification of metabolites and structurally related, presumably "tailor-made", analogs potentially designed for doping purposes.