Effect of different semiochemicals blends on spruce bark beetle,Ips typographus (Coleoptera: Scolytidae)

The spruce bark beetle, Ips typographus, is a recent new introduction to the Qilian Mountains of China. An outbreak of these beetles has infested over 0.03 million hectares of spruce forests in this area. Although primary attraction to volatiles has been clearly demonstrated for I. typographus, the existence and role of attraction to insect‐produced pheromones have been widely debated. Currently, commercial lures for I. typographus include only the volatiles ipsdienol, cis‐verbenol, trans‐verbenol, 2‐methyl‐3‐buten‐2‐ol and 2‐phenylethanol in Europe. Several potential pheromone candidates have been identified for I. typographus. Our GC–MS and GC–FID analyses volatiles from hindgut extracts of I. typographus in different attack phases demonstrated that the 2‐methyl‐3‐buten‐2‐ol, ipsdienol, cis‐verbenol and trans‐verbenol as major hindgut components, and ipsenol, 2‐phenylethanol, trans‐ myrtenol and verbenone as minor components. We tested various combinations of semiochemical candidates, to determine an optimal blend. Our results suggest that addition of 2‐methyl‐3‐buten‐2‐ol to either ipsenol alone, or to blends of ipsenol and other semiochemical candidates, significantly enhanced attraction of I. typographus. Therefore, a simple lure consisting of ipsenol and 2‐methyl‐3‐buten‐2‐ol would be an optimal blend of I. typographus in the Qilian Mountains, China. We conclude that this optimal semiochemical blend may provide an effective biological pest control method for use in forest ecosystem against I. typographus.     

Lipidomic Analysis Reveals Altered Fatty Acid Metabolism in the Liver of the Symptomatic Niemann–Pick,Type C1 Mouse Model

Niemann–Pick disease, type C1 (NPC1) is a fatal, autosomal recessive, neurodegenerative disorder caused by mutations in the NPC1 gene. As a result of the genetic defect, there is accumulation of unesterified cholesterol and sphingolipids in the late endosomal/lysosomal system causing both visceral and neurological defects. These manifest clinically as hepatosplenomegaly, liver dysfunction, and neurodegeneration. While significant progress has been made to better understand NPC1, the downstream effects of cholesterol storage and the major mechanisms that drive these pathologies remains less understood. In this study, it is sought to investigate free fatty acid levels in Npc1^?/? mice with focus on the polyunsaturated ω‐3 and ω‐6 fatty acids. Since fatty acids are the main constituents of numerous lipids species, a discovery based lipidomic study of liver tissue in Npc1^?/? mice is also performed. To this end, alterations in fatty acid synthesis, including the ω‐3 and 6 fatty acids, are reported. Further, alterations in enzymes that regulate the synthesis of ω‐3 and 6 fatty acids are reported. Analysis of the liver lipidome reveals alterations in both storage and membrane lipids including ceramides, fatty acids, phosphatidylcholamines, phosphatidylglycerols, phosphatidylethanolamines, sphingomyelins, and triacylglycerols in Npc1^?/? mice at a late stage of disease.     

hmSEEKER: Identification of hmSILAC Doublets in MaxQuant Output Data

Heavy methyl Stable Isotope Labeling with Amino acids in Cell culture (hmSILAC) is a metabolic labeling strategy employed in proteomics to increase the confidence of global identification of methylated peptides by MS. However, to this day, the automatic and robust identification of heavy and light peak doublets from MS‐raw data of hmSILAC experiments is a challenging task, for which the choice of computational methods is very limited. Here, hmSEEKER, a software designed to work downstream of a MaxQuant analysis for in‐depth search of MS peak pairs that correspond to light and heavy methyl‐peptide within MaxQuant‐generated tables is described with good sensitivity and specificity. The software is written in Perl, and its code and user manual are freely available at Bitbucket ( https://bit.ly/2scCT9u ).     

Excretion of corticosteroids in urine and faeces of hares (Lepus europaeus)

Increased production of glucocorticoids by the adrenal cortex is found in mammals under stress. As cortisol itself is absent in the faeces, an enzyme immunoassay (11-oxoaetiocholanolone) measuring 11,17-dioxoandrostanes has already been established to measure faecal cortisol metabolites in ruminants for non-invasive monitoring of adrenocortical activity. The aim of this study was to establish route and delay of excretion of glucocorticoids in hares and to determine whether a cortisol-, corticosterone- or this new enzyme immunoassay is best suited to detect faecal glucocorticoid metabolites. In the first experiment radioactive-labelled glucocorticoids (¹⁴C-cortisol and ³H-corticosterone) were administered intravenously to two groups of three hares in metabolic cages. All voided urine and faecal samples were collected for 4 days. Metabolites of both steroids were found predominantly in the urine (91 ± 4%). Peak concentrations were observed in the first urinary sample following infusion (13 ± 6 h) and in the faeces with a delay of about 1 day (23 ± 7 h). Most of the radioactivity was not extractable with diethylether, indicating that the metabolites excreted in urine and faeces are mainly conjugated or polar unconjugated ones. This was confirmed by reverse-phase high-performance liquid chromatography separations of the metabolites, which also revealed marked differences concerning the metabolism of the two glucocorticoids injected. Compared with the cortisol and the corticosterone enzyme immunoassay, only the group-specific enzyme immunoassay for 11,17-dioxoandrostanes detected high quantities of immunoreactive metabolites. In a second experiment hares (n=20) were stressed by rousing them three times (5 min, 10 min and another 5 min) with a 20-min break in-between. Faecal samples were collected 2 days before until 4 days after stress and analysed using the 11-oxoaetiocholanolone enzyme immunoassay. After stress significantly (P < 0.001) increased 11,17-dioxoandrostane concentrations were found. Based on these results, measuring 11,17-dioxoandrostanes in faeces enables non-invasive monitoring of disturbances in hares and thus provides a tool for field investigations elucidating the role of stress in hare populations. Accepted: 24 November 1999     

Measurement of endogenous Na+,K+-ATPase inhibitors in human plasma and urine using high-performance liquid chromatography

This study was undertaken to assess endogenous Na+,K+-ATPase inhibitors in both plasma and urine in the same subjects. Samples were chromatographed on reverse-phase HPLC using an acetonitrile gradient and the eluent screened using Na+,K+-ATPase inhibition and cross-reaction with anti-digoxin antibodies. The donors were divided into inhibiting and non-inhibiting subjects using a previously described method, plasma action on ouabain binding and on Na+,K+-ATPase activity. Three Na+,K+-ATPase inhibitors (1P, 2P and 3P) were detectable in plasma; the antibodies cross-reaction of the peaks 2P and 3P were larger than that of peak 1P. The peaks 2P and 3P were significantly higher in inhibiting subjects as compared to non-inhibiting subjects. The 24-h urine is resolved into two peaks inhibiting Na+,K+-ATPase activity (1U and 2U). Peak 2U cross-reacted with anti-digoxin antibodies to a greater extent than peak 1U and is significantly larger in inhibiting subjects in terms of Na+,K+-ATPase inhibition. These data support the heterogeneity of human Na+,K+-ATPase inhibitor in both plasma and urine.     

Active and inactive metabolic pathways in tumor spheroids: Determination by GC–MS

Active metabolic pathways in three‐dimensional cancer‐cell cultures are potential chemotherapeutic targets that would be effective throughout tumors. Chaotic vasculature creates cellular regions in tumors with distinct metabolic behavior that are only present in aggregate cell masses. To quantify cancer cell metabolism, transformed mouse fibroblasts were grown as spheroids and fed isotopically labeled culture medium. Metabolite uptake and production rates were measured as functions of time. Gas chromatography–mass spectrometry was used to quantify the extent of labeling on amino acids present in cytoplasmic extracts. The labeling pattern identified several active and inactive metabolic pathways: Glutaminolysis was found to be active, and malic enzyme and gluconeogenesis were inactive. Transformed cells in spheroids were also found to actively synthesize serine, cysteine, alanine, aspartate, glutamate, and proline; and not synthesize glutamine. The activities of these pathways suggest that cancer cells consume glutamine for biosynthesis and not to provide cellular energy. Determining active metabolic pathways indicates how cells direct carbon flow and may lead to the discovery of novel molecular targets for anticancer therapy. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Determination of phenethyl isothiocyanate in human plasma and urine by ammonia derivatization and liquid chromatography-tandem mass spectrometry

Phenethyl isothiocyanate (PEITC) is a dietary compound present in cruciferous vegetables that has cancer-preventive properties. Our objective was to develop and validate a novel liquid chromatography-tandem mass spectrometry procedure to analyze PEITC concentrations in human plasma and urine. Following hexane extraction, ammonia was added to samples to derivatize PEITC to phenethylthiourea. Chromatographic separation was achieved on a C(18) column with acetonitrile/5 mM formic acid (60:40, v/v) as the mobile phase followed by tandem mass spectrometry detection in multiple reaction monitoring mode. Deuterium-labeled PEITC was used as the internal standard. The detection limit was 2 nM and calibration curves were linear from 7.8 to 2000 nM. The intra- and inter-day coefficients of variation were less than 5 and 10%, respectively. The intra- and inter-day accuracies ranged from 101.0 to 104.2% and from 102.8 to 118.6%, respectively. The recovery from spiked human plasma and urine ranged from 100.3 to 113.5% and from 98.3 to 103.9%, respectively. The assay was used to measure PEITC in plasma and urine samples obtained from subjects after consumption of 100g of watercress. This novel assay represents the first analytical method with the sensitivity and specificity to determine plasma and urine concentrations of PEITC.     

Comparative study of liquefaction process and liquefied products from bamboo using different organic solvents

The effects of various solvents, including phenol, ethylene glycol (EG) and ethylene carbonate (EC), and different liquid ratios on the liquefaction of bamboo, have been studied systematically in this paper. The processes were catalyzed by hydrochloride acid at 180 °C in autoclaves for different reaction times. The results show that phenol is the optimum solvent for bamboo liquefaction with a yield up to 99%. The Fourier transform-infrared (FT-IR) analyses of the residues show that cellulose, hemicelluloses and lignin are almost decomposed when using phenol as solvent. The gel permeation chromatography (GPC) results of the liquid products show that the high molecular weight of bamboo decreases significantly to around 1800 g mol⁻¹ after liquefaction. The gas chromatography and mass spectrometry (GC–MS) analysis shows that low boiling point products of liquefied bamboo are similar regardless of the type of solvent used.     

Composition and emission dynamics of migratory locust volatiles in response to changes in developmental stages and population density

Chemical communication plays an important role in density‐dependent phase change in locusts. However, the volatile components and emission patterns of the migratory locust, Locusta migratoria, are largely unknown. In this study, we identified the chemical compositions and emission dynamics of locust volatiles from the body and feces and associated them with developmental stages, sexes and phase changes. The migratory locust shares a number of volatile components with the desert locust (Schistocerca gregaria), but the emission dynamics of the two locust species are significantly different. The body odors of the gregarious nymphs in the migratory locust consisted of phenylacetonitrile (PAN), benzaldehyde, guaiacol, phenol, aliphatic acids and 2,3‐butanediol, and PAN was the dominant volatile. Volatiles from the fecal pellets of the nymphs primarily consist of guaiacol and phenol. Principal component analysis (PCA) showed significant differences in the volatile profiles between gregarious and solitary locusts. PAN and 4‐vinylanisole concentrations were significantly higher in gregarious individuals than in solitary locusts. Gregarious mature males released significantly higher amounts of PAN and 4‐vinylanisole during adulthood than mature females and immature adults of both sexes. Furthermore, PAN and 4‐vinylanisole were completely lost in gregarious nymphs during the solitarization process, but were obtained by solitary nymphs during gregarization. The amounts of benzaldehyde, guaiacol and phenol only unidirectionally decreased from solitary to crowded treatment. Aliphatic aldehydes (C7 to C10), which were previously reported as locust volatiles, are now identified as environmental contaminants. Therefore, our results illustrate the precise odor profiles of migratory locusts during developmental stages, sexes and phase change. However, the function and role of PAN and other aromatic compounds during phase transition need further investigation.     

Pitfalls in histone propionylation during bottom‐up mass spectrometry analysis

Despite their important role in regulating gene expression, posttranslational histone modifications remain technically challenging to analyze. For identification by bottom‐up MS, propionylation is required prior to and following trypsin digestion. Hereby, more hydrophobic peptides are generated enabling RP HPLC separation. When histone dynamics are studied in a quantitative manner, specificity, and efficiency of this chemical derivatization are crucial. Therefore we examined eight different protocols, including two different propionylation reagents. This revealed amidation (up to 70%) and methylation (up to 9%) of carboxyl groups as a side reaction. Moreover, incomplete (up to 85%) as well as a specific propionylation (up to 63%) can occur, depending on the protocol. These results highlight the possible pitfalls and implications for data analysis when doing bottom‐up MS on histones.     

Terpenes associated with resistance against the gall wasp,Leptocybe invasa,in Eucalyptus grandis

Leptocybe invasa is an insect pest causing gall formation on oviposited shoot tips and leaves of Eucalyptus trees leading to leaf deformation, stunting, and death in severe cases. We previously observed different constitutive and induced terpenes, plant specialized metabolites that may act as attractants or repellents to insects, in a resistant and susceptible clone of Eucalyptus challenged with L. invasa. We tested the hypothesis that specific terpenes are associated with pest resistance in a Eucalyptus grandis half‐sib population. Insect damage was scored over 2 infestation cycles, and leaves were harvested for near‐infrared reflectance (NIR) and terpene measurements. We used Bayesian model averaging for terpene selection and obtained partial least squares NIR models to predict terpene content and L. invasa infestation damage. In our optimal model, 29% of the phenotypic variation could be explained by 7 terpenes, and the monoterpene combination, limonene, α‐terpineol, and 1,8‐cineole, could be predicted with an NIR prediction ability of  .67. Bayesian model averaging supported α‐pinene, γ‐terpinene, and iso‐pinocarveol as important for predicting L. invasa infestation. Susceptibility was associated with increased γ‐terpinene and α‐pinene, which may act as a pest attractant, whereas reduced susceptibility was associated with iso‐pinocarveol, which may act to recruit parasitoids or have direct toxic effects.     

Comparative proteomic analysis of trophoblast cell models reveals their differential phenotypes,potential uses,and limitations

Trophoblastic cell lines are widely used in in vitro studies of placental function as a surrogate for primary trophoblasts. To date, no reference proteomics dataset exists to directly compare the shared and unique characteristics of these cells. Here, we performed comparative proteomic profiling of the BeWo and HTR8/SVneo cell lines using label‐free quantitative MS. A total of 1557 proteins were identified, which included 338 uniquely attributed to BeWo cells, and a further 304 specifically identified in HTR8/SVneo cells. Raw data are available via ProteomeXchange, identifier PDX005045. Of the 915 proteins expressed by both cell lines, 105 were of higher abundance in BeWo cells, while 199 proteins had a significantly higher expression in HTR8/SVneo cells. Comparative GO of unique and upregulated proteins revealed principal differences in cell junction/adhesion, catenin complex, spindle and microtubule associated complex, as well as cell differentiation. Our data indicate that BeWo cells express an epithelial proteome more characteristic of villous trophoblasts, whereas HTR8/SVneo cells embrace a mesenchymal phenotype, more characteristic of extravillous trophoblasts. This novel comparative proteomic profiling of these trophoblastic cell lines provides a useful platform for future investigations of placental function.     

Analysis of amphetamines and metabolites in urine with ultra performance liquid chromatography tandem mass spectrometry

A simple, rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry method was developed and fully validated for the quantitative determination of seven amphetamines and metabolites in urine. The method was validated for selectivity, linearity, LOQ, LOD, imprecision, bias, analyte and processed sample stability, matrix effect, recovery, carryover and dilution integrity. A classic liquid–liquid extraction with ethyl acetate was used as sample preparation procedure. The compounds were separated on an Acquity UPLC HSS C₁₈ column in 6.8 min. The linear dynamic range was established from 25 to 500 ng/mL. The limit of quantification was fixed to the lowest calibrator level and the limit of detection ranged from 0.125 to 2.5 ng/mL. The method presented an excellent intra- and inter-assay imprecision and bias (<10.7%) at each measured concentration of two external quality controls (QC) and three “in house” QC. No matrix effects were observed and good recoveries (>70%) were obtained for all the compounds. No carryover was observed after the analysis of high concentrated samples (8000 ng/mL). The method was subsequently applied to authentic samples.     

Inhibition of the growth and development of mosquito larvae of Culex pipiens L. (Diptera: Culicidae) treated with extract from flower of Matricaria chamomilla (Asteraceae)

Mosquitoes can transfer many adverse diseases to human and animals therefore, there is a need to fight their spread. Among promising larvicidal sources is the use of plant extracts, which will play an important role in the future. This study was conducted to assess the larvicidal activity of Ferula hermonis Boiss,Achillea millefolium, Salvia officnalis, Psidium guaja and Matricaria chamomilla extract against Culex pipiens. The plant materials were extracted with hexane, dichloromethane, ethyl acetate, and methanol using a Soxhlet extractor. The extracts were evaluated for larvicidal activity against Cx. pipiens the mortality was monitored after 24 and 48 h of exposure. None of the extracts tested showed larvicidal acitivties except M. chamomilla. The ethyl acetate extract showed the most promising larvicidal activity with LC₅₀ values of 287.1 and 209.4 ppm after 24 and 48 h of exposure, respectively. Treatment of the eggs with different concentrations of the active extract decreased the hatchability of the eggs dose dependently from 95 to 86.49%. Similarly, the pupal duration increased in treated groups. The larval period lasted for 12 d, whereas that of the control group lasted for 10 d. Furthermore, the pupal period lasted 3 d (control 2 d) in treated groups. The data also revealed a significant decrease in the growth index in treated groups (0.00–7.53) than that of the control (8.5). The GC–MS analysis revealed the presence of 1.6.10‐dodecatriene, 7,11‐dimethyl‐3‐methylene (89.68%), 1,6‐cyclodecadiene, 1‐methyl‐5‐methylene‐8‐(1‐methylethyl)‐, [S‐(E,E)] (6.34%), 2H‐pyran ?3‐ol (4.04%), and 2H‐1‐benzopyran‐2‐one (0.079%).     

Polar Constituents of the Tunicate Botryllus schlosseri

Eighteen compounds were identified by GC–MS of their trimethylsilyl derivatives in n-butanolic extract from the biomass of Botryllus schlosseri. Three of them, 5-oxoproline, 5-hydroxyhydantoin, and kinurenic acid, were found in marine invertebrates for the first time. In addition to cellulose, the biomass was also shown to contain complex water-soluble sulfated polysaccharides. These were extracted and fractionated, and sulfate content and monosaccharide composition were determined in the fractions; fucose, xylose, galactose, mannose, glucose, glucosamine, galactosamine, and uronic acids were found. Unlike several other tunicate species, Botryllus schlosseri does not seem to contain any simple galactan sulfate.     

Variation of metabolites in normal human urine

Urine is often sampled from patients participating in clinical and metabolomic studies. Biological homeostasis occurs in humans, but little is known about the variability of metabolites found in urine. It is important to define the inter- and intra-individual metabolite variance within a normal population before scientific or clinical conclusions are made regarding different pathophysiologies. This study investigates the variability of selected urine metabolites in a group of 60 healthy men and women over a period of 30 days. To monitor individual variation, 6 women from the normal population were randomly selected and followed for 30 days. To determine the influence of extraneous environmental factors urine was collected from 25 guinea pigs with similar genetics, diet, and living environment. For both studies, 24 metabolites were identified and quantified using high-resolution ¹H nuclear magnetic resonance spectroscopy (NMR). The data demonstrated large inter and intra-individual variation in metabolite concentrations in both normal human and control animal populations. A defined normal baseline is essential before any conclusions may be drawn regarding changes in urine metabolite concentrations.