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In the light of the current discussion on reduced insect biomass and species decline, we would like to draw attention to the work of amateur entomologists who have been observing the moth and butterfly fauna for decades. Actually, the recording of butterflies and moths has a long tradition in Wuppertal and its surroundings (Germany, North Rhine-Westfalia, Bergisches Land). Therefore, we have access to rather detailed data of the local macrolepidoptera fauna collected over the last 160 years and are able to comment on the trends of moth and butterfly populations during this rather long period. We review historical and current data and provide a comprehensive abundance list of all macrolepidoptera species observed in the study region. We found that, from the mid-twentieth century onwards, the species richness of butterfly and moths species decreased considerably. In terms of the number of species evaluated (537), we see that 27% decreased within the last 160 years while 15% have already been lost. Additionally, 24% are apparently stable at a low level. Particularly affected are highly specialised species of heath, moor, grassland, scrub, coppice and orchard habitats. However, 15% of the evaluated species are observed more frequently. Some of these newly colonised the study region (2.4%). Since Wuppertal is a city that profited from the industrial revolution from the middle of the nineteenth century onwards, we think that our results could serve as a representative example of the loss of species richness due to industrialisation, urbanisation, intensive agriculture and forestry. If we intend to increase species richness of butterflies and moths again, the focus must be on protecting, restoring and promoting low-nutrient open landscape habitats rather than forests.

Detecting agents allegedly or evidently promoting growth such as human growth hormone (GH) or growth hormone releasing peptides (GHRP) in doping controls has represented a pressing issue for sports drug testing laboratories. While GH is a recombinant protein with a molecular weight of 22 kDa, the GHRPs are short (3–6 amino acids long) peptides with GH releasing properties. The endogenously produced GH (22 kDa isoform) consists of 191 amino acids and has a monoisotopic molecular mass of 22,124 Da. Within this study, a slightly modified form of GH was discovered consisting of 192 amino acids carrying an additional alanine at the N-terminus, leading to a monoisotopic mass of 22,195 Da. This was confirmed by top-down and bottom-up experiments using liquid chromatography coupled to high resolution/high accuracy mass spectrometry. Additionally, three analogues of GHRPs were identified as Gly-GHRP-6, Gly-GHRP-2 and Gly-Ipamorelin, representing the corresponding GHRP extended by a N-terminal glycine residue. The structure of these peptides was characterised by means of high resolution (tandem) mass spectrometry, and for Gly-Ipamorelin and Gly-GHRP-2 their identity was additionally confirmed by custom synthesis. Further, established in-vitro experiments provided preliminary information considering the potential metabolism after administration.

Even without clinical approval, many performance-enhancing drugs are available on the black market and can therefore be easily obtained by cheating athletes. The misuse of these preparations can be associated with unforeseeable health risks - either due to a poor quality of the drugs or as a result of an insufficient clinical assessment. Moreover, confiscated black market products have frequently been shown to contain ingredients other than those declared on the label as well as additional by-products or compounds with a modified molecular structure. This communication describes the identification of an unknown fusion protein observed in several unlabelled black market products obtained from independent sources. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the confiscated preparations indicated the presence of an 18-kDa fusion protein consisting of the bacterial redox protein thioredoxin-1 (Trx, 12 kDa) and a 6-kDa peptide of unassigned composition. Trx has no relevance as performance enhancing agent but is routinely used as solubility tag for recombinant protein production. Further evaluation of the acquired MS/MS data revealed both an additional His tag and a thrombin cleavage site between the tags and the presumed bioactive peptide. However, thrombin cleavage of the fusion protein and LC-MS/MS analysis of the resulting peptide fragment finally suggested that the unknown protein is only the product of an empty expression vector without the DNA insert of interest. These findings are a further alarming example for the high level of risk that athletes take when misusing drugs obtained from the black market. Copyright © 2014 John Wiley & Sons, Ltd. Language: en

The chemical composition of a black powder confiscated by German customs was elucidated. Black powders are occasionally used as a 'transporter' for cocaine and are obviously especially designed to cloak the presence of the drug. The material consisting of cocaine, copper, iron, thiocyanate, and graphite was approached by analytical tools and chemical modelling. Graphite is added to the material probably with the intention of masking the typical infrared (IR) fingerprints of cocaine and can be clearly detected by powder X-ray diffraction (XRD) and Raman spectroscopy. Cu(2+) and NCS(-) ions, when carefully reacted with cocaine hydrochloride, form the novel compound (CocH)2 [Cu(NCS)4 ] (CocH(+) = protonated cocaine), which has been characterised by single crystal XRD, IR, NMR, UV/Vis absorption and EPR spectroscopy. Based on some further experiments the assumed composition of the original black powder is discussed. Copyright © 2014 John Wiley & Sons, Ltd. Language: en

Six collected phenidates, i.e., 4-methylmethylphenidate, 3,4-dichloromethylphenidate, ethylphenidate, 3,4-dichloroethylphenidate, ethylnaphthidate, and N-benzyl-ethylphenidate were fully characterized by means of X-ray, nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS), electrospray ionization-tandem mass spectrometry (ESI-MS/MS), attenuated total reflectance Fourier transform infrared (ATR-FTIR) and GC solid-state IR analysis. Crystallography revealed the exclusive presence of the threo-configuration. Steric crowding induced by N-benzyl substitution at the piperidine moiety prompted an adoption of an unexpected axial positioning of substituents on the piperidine moiety in the crystal state as opposed to the exclusive equatorial positioning encountered in N-unsubstituted phenidate analogues. Gas phase computations of the relative lowest energy conformers confirm that the axial positioning appears to be favoured over the equatorial positioning; in solution, however, equatorial positioning is predominant according to nuclear Overhauser effect experiments. All samples, mainly originating from China, had a good to very good degree of purity indicative of their professional chemical synthesis. Routine analysis of these drugs by GC-MS revealed thermal decomposition of phenidate analogues in the injection port and/or on column to 2-aryl-ethyl-acetates and 2,3,4,5-tetrahydropyridines. The decomposition pathway was suggested to proceed via a 6-membered transition state which was supported by density functional theory (DFT) computations. Fragmentation pathways of decomposition products as well as the corresponding electron ionization (EI) mass spectra are provided. The thermal instability might thus render smoking or 'vaping' of these drugs a less effective route of administration. The analytical fingerprints of six structurally diverse phenidate analogues provide a helpful reference to forensic chemists in charge of identifying new psychoactive substances. Copyright © 2017 John WileySons, Ltd.

The use of growth factors for accelerated healing of sports injuries is restricted under the terms of the World Anti-Doping Agency (WADA) anti-doping code. Cheating athletes have used the black market as a source of performance-enhancing substances. Drugs that currently undergo clinical trials are frequently offered – despite the unknown health risks associated with the administration of unapproved pharmaceuticals. Recently, a new growth factor (referred to as fibroblast growth factor 1/ FGF-1) with known effects on the repair and regeneration of damaged tissue was detected in an unlabelled black market product confiscated by the German customs. The identification of the protein was achieved by one- and two-dimensional polyacrylamide gel electrophoresis (SDS-PAGE and 2D-PAGE), different proteolytic digestions, immunological methods and nano-liquid chromatography high-resolution/high-accuracy Orbitrap mass spectrometry. The SDS-PAGE analysis revealed slight differences concerning the molecular weight of recombinant human and black market FGF-1. Using in-gel proteolysis, a truncation or modification located at the N-terminus of the protein was suggested. These findings demonstrate that drug candidates without clinical approval can be readily obtained from the black market, regardless of potential dangerous consequences for the consumer, which corroborates the necessity of proactive and preventive doping control approaches. In that regard, physiological concentrations of blood and urine specimens collected from healthy individuals were analyzed and were found to range below 28pg/ml in urine, while there was no detectable FGF-1 in plasma. Copyright © 2011 John Wiley & Sons, Ltd.

The desire to increase the athletic performance, to ‘optimize’ an individual’s appearance, and to complement but also to arguably substitute exercise by means of drugs and drug candidates has generated a considerable (illicit) market for compounds such as anabolic-androgenic steroids, stimulants, growth promoting peptide hormones, and so on. Genuinely developed for therapeutic use, their abuse/misuse generates enormous health risks, which has necessitated comprehensive controls of compound trafficking by customs and anti-doping authorities. From 2012 to 2013, the Bureau of Customs Investigation confiscated products containing anabolic-androgenic steroids (AAS; 259 kg), stimulants (13 kg), selective estrogen receptor modulators (SERMs; 24 kg), and human growth hormone (hGH; 3500 ampules). In cooperation with the Bureau and under the umbrella of the European Monitoring Center for Emerging Doping Agents (EuMoCEDA), the Cologne Anti-Doping Laboratory analyzed an additional 337 (black market) products between 2010 and 2013, allowing to monitor developments in drug use and, hence, the anticipation of new challenges in sports drug testing. Main tools utilized in characterizing confiscated materials were liquid chromatography-high resolution mass spectrometry (LC-HRMS), gas chromatography-high resolution mass spectrometry (GC-HRMS), and polyacrylamide gel electrophoresis (PAGE) with subsequent bottom-up identification of peptidic compounds using nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). Among the 337 substances analyzed in the doping control laboratory in Cologne, 67 active ingredients were found, 49 of which being categorized as doping agents by the World Anti-Doping Agency (WADA). A total of 83.7 % accounted for steroidal substances (predominantly testosterone, trenbolone, and nandrolone and corresponding esters), 12.8 % accounted for peptide hormones and growth factors (predominantly hGH and growth hormone releasing peptides (GHRPs)), 3.2 % of the products contained hormones and metabolic modulators, and 0.3 % accounted for diuretic agents. Outstanding findings were the detection of the selective androgen receptor modulator (SARM) LGD-4033, the thymic hormone thymosin β4, and a fusion protein of unknown biological activity. Trafficking of considerable amounts of arguably performance and/or body-enhancing compounds has been observed during the past 4 years, the majority of which is categorized as relevant to sports drug testing. Several substances are of fake/non-approved nature and represent enormous health risks to the ‘customer’.

Even without clinical approval, many performance-enhancing drugs are available on the black market and can therefore be easily obtained by cheating athletes. The misuse of these preparations can be associated with unforeseeable health risks - either due to a poor quality of the drugs or as a result of an insufficient clinical assessment. Moreover, confiscated black market products have frequently been shown to contain ingredients other than those declared on the label as well as additional by-products or compounds with a modified molecular structure. This communication describes the identification of an unknown fusion protein observed in several unlabelled black market products obtained from independent sources. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the confiscated preparations indicated the presence of an 18-kDa fusion protein consisting of the bacterial redox protein thioredoxin-1 (Trx, 12 kDa) and a 6-kDa peptide of unassigned composition. Trx has no relevance as performance enhancing agent but is routinely used as solubility tag for recombinant protein production. Further evaluation of the acquired MS/MS data revealed both an additional His tag and a thrombin cleavage site between the tags and the presumed bioactive peptide. However, thrombin cleavage of the fusion protein and LC-MS/MS analysis of the resulting peptide fragment finally suggested that the unknown protein is only the product of an empty expression vector without the DNA insert of interest. These findings are a further alarming example for the high level of risk that athletes take when misusing drugs obtained from the black market.

The chemical composition of a black powder confiscated by German customs was elucidated. Black powders are occasionally used as a 'transporter' for cocaine and are obviously especially designed to cloak the presence of the drug. The material consisting of cocaine, copper, iron, thiocyanate, and graphite was approached by analytical tools and chemical modelling. Graphite is added to the material probably with the intention of masking the typical infrared (IR) fingerprints of cocaine and can be clearly detected by powder X-ray diffraction (XRD) and Raman spectroscopy. Cu(2+) and NCS(-) ions, when carefully reacted with cocaine hydrochloride, form the novel compound (CocH)2 [Cu(NCS)4 ] (CocH(+) = protonated cocaine), which has been characterised by single crystal XRD, IR, NMR, UV/Vis absorption and EPR spectroscopy. Based on some further experiments the assumed composition of the original black powder is discussed.

We have reexamined the structure of inositol phosphates present in trophozoites of the parasitic amoeba Entamoeba histolytica and show here that, rather than being myo-inositol derivatives (Martin, J.-B., Bakker-Grunwald, T., and Klein, G. (1993) Eur. J. Biochem. 214, 711-718), these compounds belong to a new class of inositol phosphates in which the cyclitol isomer is neo-inositol. The structures of neo-inositol hexakisphosphate, 2-diphospho-neo-inositol pentakisphosphate, and 2, 5-bisdiphospho-neo-inositol tetrakisphosphate, which are present in E. histolytica at concentrations of 0.08-0.36 mM, were solved by two-dimensional (31)P-(1)H NMR spectroscopy. No evidence for the co-existence of their myo-inositol counterparts has been found. These neo-inositol compounds were not substrates of 6-diphospho-inositol pentakisphosphate 5-kinase, an enzyme purified from Dictyostelium discoideum that phosphorylates 6-diphospho-myo-inositol pentakisphosphate and more slowly also myo-inositol hexakisphosphate, specifically on position 5. Because preliminary data indicate that large amounts of the same neo-inositol phosphate and diphosphate esters are also present in another primitive amoeba, Phreatamoeba balamuthi, the occurrence of high concentrations of neo-inositol polyphosphates may be much more general than previously thought.

The intracellular amounts of diphospho-myo-inositol phosphates and InsP6 were determined in Dictyostelium discoideum AX2 throughout the life cycle, including exponential growth, starvation, differentiation, sporulation and spore germination. Similar experiments were performed with the closely related species Polysphondylium pallidum under conditions resulting in microcyst formation. A distinct accumulation of these compounds is observed during the early starvation phase of the cell population before the onset of the actual differentiation program. When exponentially growing D. discoideum cells were shifted to starvation conditions, a 25-fold accumulation of 5,6-bis-PP-InsP4 within 3 h was observed. In P. pallidum, the 5,6-bis-PP-InsP4 pool rises around 20-fold within 8 h during the formation of microcysts from vegetative cells. Finally, the diphosphoinositol phosphates are deposited in spores or microcysts and are degraded when spores or microcysts germinate at low cell density.

Diphospho-myo-inositol phosphates (PP-InsP5 and bis-PP-InsP4) were isolated from Dictyostelium in order to clarify the precise positional isomerism by two-dimensional 1H/31P-NMR analysis. The diphosphorylated inositol phosphates are 4-PP-Ins(1,2,3,5,6)P5 and 4,5-bis-PP-Ins(1,2,3,6)P4 or their corresponding enantiomers. The vicinal arrangement of the diphospho groups with its steric and electrostatic constraints possibly qualifies bis-PP-InsP4 as a metabolite with high phosphate-group-transfer potential in phosphotransferase reactions.

In the heart, in situ local myocardial blood flow (MBF) varies greater than 10-fold between individual areas and displays a spatially heterogeneous pattern. To analyze its molecular basis, we analyzed protein expression of low and high flow samples (300 mg, 150% of mean MBF, each n=30) of six beagle dogs by 2-D polyacrylamide gel electrophoresis (380 +/- 78 spots/gel). In low flow samples, dimethylarginine dimethylaminohydrolase (DDAH1) was increased greatly (+377%, compared with high flow samples). This increase resulted in a 75% reduction of asymmetric dimethylarginine (ADMA), the potent endogenous inhibitor of NO synthase, whereas eNOS showed no difference. Low flow samples exhibited enhanced expression of GAPDH (+89%) and phosphoglycerate kinase (+100%), whereas hydroxyacyl-CoA dehydrogenase, electron transfer flavoprotein, myoglobin, and desmin were decreased. Assessing local MBF on different days within 2 weeks revealed a high degree of MBF stability (r2 > 0.79). Thus, stable differences in local MBF are associated with significant differences in local gene and protein expression. In low flow areas, the increased DDAH1 reduces ADMA concentration and NOS inhibition, which strongly suggests enhanced NO formation. Low flow areas are also characterized by a higher glycolytic and a lower fatty acid oxidation capacity. Both the shift in substrate utilization and the rise in NO may contribute to the known lower oxygen consumption in these areas

The use of growth factors for accelerated healing of sports injuries is restricted under the terms of the World Anti-Doping Agency (WADA) anti-doping code. Cheating athletes have used the black market as a source of performance-enhancing substances. Drugs that currently undergo clinical trials are frequently offered--despite the unknown health risks associated with the administration of unapproved pharmaceuticals. Recently, a new growth factor (referred to as fibroblast growth factor 1/FGF-1) with known effects on the repair and regeneration of damaged tissue was detected in an unlabelled black market product confiscated by the German customs. The identification of the protein was achieved by one- and two-dimensional polyacrylamide gel electrophoresis (SDS-PAGE and 2D-PAGE), different proteolytic digestions, immunological methods and nano-liquid chromatography high-resolution/high-accuracy Orbitrap mass spectrometry. The SDS-PAGE analysis revealed slight differences concerning the molecular weight of recombinant human and black market FGF-1. Using in-gel proteolysis, a truncation or modification located at the N-terminus of the protein was suggested. These findings demonstrate that drug candidates without clinical approval can be readily obtained from the black market, regardless of potential dangerous consequences for the consumer, which corroborates the necessity of proactive and preventive doping control approaches. In that regard, physiological concentrations of blood and urine specimens collected from healthy individuals were analyzed and were found to range below 28 pg/ml in urine, while there was no detectable FGF-1 in plasma.

Hallucinogenic mushrooms (e.g. Psilocybe and Panaeolus species) as well as leaves and young shoots of the khat tree (Catha edulis Forsk) are illicit drugs in many countries. The exact concentration of the hallucinogenic alkaloids psilocin and psilocybin in mushrooms and the sympathomimetic alkaloids cathinone and cathine in khat is usually essential for jurisdiction. Facing an increasing number of mushroom and khat seizures by German customs authorities, a convenient comprehensive quantitative HPLC method based on cation-exchange liquid chromatography for these rather "exotic" drugs has been developed which avoids time-consuming multi-step sample preparation or chemical derivatization procedures. Using this method a number of different hallucinogenic fungi species and products that are mainly distributed via the internet have been analysed (dried and fresh Psilocybe cubensis Singer as well as P. cubensis collected from "grow boxes", Panaeolus cyanescens Berkeley and Broome and so-called "philosopher stones" (sclerotia of Psilocybe species)). Highest total amounts of psilocin have been detected in dried P. cyanescens reaching up to 3.00+/-0.24 mg per 100 mg. The distribution of khat alkaloids in different parts of the khat shoots has been studied. High concentrations of cathinone have not only been detected in leaves but also in green parts and barks of stalks. Additionally, the sample treatment for fresh mushroom and khat samples has been optimised. Highest amounts of alkaloids were found when fresh material was freeze-dried.

Two diphospho- myo -inositol phosphates from Dictyostelium were recently investigated by two-dimensional 1 H/ 31 P NMR analysis and assigned to be either d-4-diphospho- myo -inositol pentakisphosphate (d-4- PP -Ins P 5 ) and d-4,5-bisdiphospho- myo -inositol tetrakisphosphate (d-4,5-bis- PP -Ins P 4 ) or their corresponding enantiomers d-6- PP -Ins P 5 and d-5,6-bis- PP -Ins P 4 . In the present study the naturally occurring enantiomers were identified by using defined synthetic PP -Ins P 5 isomers as substrates for a partially purified PP -Ins P 5 5-kinase from Dictyostelium . This enzyme specifically phosphorylates the naturally occurring PP -Ins P 5 and the synthetic d-6- PP -Ins P 5 , leading to d-5,6-bis- PP -Ins P 4 . In contrast, neither d-4- PP -Ins P 5 nor d-1- PP -Ins P 5 or d-3- PP -Ins P 5 are converted by the enzyme.

Myoglobin is an important intracellular O 2 binding hemoprotein in heart and skeletal muscle. Surprisingly, disruption of myoglobin in mice (myo −/− ) resulted in no obvious phenotype and normal cardiac function was suggested to be mediated by structural alterations that tend to steepen the oxygen pressure gradient from capillary to mitochondria. Here we report that lack of myoglobin causes a biochemical shift in cardiac substrate utilization from fatty acid to glucose oxidation. Proteome and gene expression analysis uncovered key enzymes of mitochondrial β-oxidation as well as the nuclear receptor PPARα to be downregulated in myoglobin-deficient hearts. Using FDG-PET we showed a substantially increased in vivo cardiac uptake of glucose in myo −/− mice (6.7±2.3 versus 0.8±0.5% of injected dose in wild-type, n=5, P 13 C NMR spetroscopic isotopomer studies of isolated hearts which revealed that [1,6- 13 C 2 ]glucose utilization was increased in myo −/− hearts (38±8% versus 22±5% in wild-type, n=6, P 13 C 16 ]palmitate utilization was decreased in the myoglobin-deficient group (42±6% versus 63±11% in wild-type, n=6, P 2 -sparing effect of glucose utilization, the observed shift in substrate metabolism benefits energy homoeostasis and therefore represents a molecular adaptation process allowing to compensate for lack of the cytosolic oxygen carrier myoglobin. Furthermore, our data suggest that an altered myoglobin level itself may be a critical determinant for substrate selection in the heart. The full text of this article is available online at http://circres.ahajournals.org.

Myoglobin knockout (myo-/-) mice were previously reported to show no obvious phenotype but revealed several compensatory mechanisms that include increases in cardiac capillary density, coronary flow, and hemoglobin. The aim of this study was to investigate whether severe hypoxic stress can exhaust these compensatory mechanisms and whether this can be monitored on the gene and protein level. Myo-/- and wild-type (WT) mice were exposed to hypoxia (10% O2) for 2 wk. Thereafter hemodynamic parameters were investigated by invasive measurement combined with magnetic resonance imaging. Cardiac gene and protein expression were analyzed using cDNA arrays and two-dimensional gel electrophoresis plus mass spectrometry, respectively. Hematocrit levels increased from 44% (WT) and 48% (myo-/-) to 72% in both groups. Similar to WT controls, hypoxic myo-/- animals maintained stable cardiovascular function (mean arterial blood pressure 82.4 mmHg, ejection fraction 72.5%). Cardiac gene expression of hypoxic myo-/- mice differed significantly from WT controls in 17 genes (e.g., keratinocyte lipid binding protein +202%, cytochrome c oxidase Vb +41%). Interestingly, hypoxia inducible factor-1α remained unchanged in both groups. Proteome analysis revealed reduced levels of heart fatty acid-binding protein and heat shock protein 27 both in hypoxic myo-/- and WT mice. Our data thus demonstrate that myo-/- mice do not decompensate during hypoxic stress but are surprisingly well adapted. Changes in energy metabolism of fatty acids may contribute to the robustness of myoglobin-deficient mice.

The two major diphospho inositol phosphates from the axenic strain Dictyostelium discoideum AX2 were previously investigated and identified as 6-PP-InsP5 and 5,6-bis-PP-InsP4. In order to examine whether these findings are representative of Dictyostelids in general, five non-axenic wild-type species of Dictyostelium and two of Polysphondylium were studied. It was found that all of the Dictyostelium species exhibit similar patterns of diphospho inositol phosphates. By contrast, both of the Polysphondylium species contain 5-PP-InsP5 as the predominant isomer. Besides 5,6-bis-PP-InsP4, a new bis-PP-InsP4 was detected in Polysphondylium. This compound is either 1,5-bis-PP-InsP4 or its corresponding enantiomer 3,5-bis-PP-InsP5. The structures were elucidated by two-dimensional 1H-1H and 1H-31P NMR analysis. Additionally, they were confirmed using a specific 6-PP-InsP5-5-kinase from D. discoideum AX2 as an enantio-specific tool and enantiomerically pure reference standards.