A novel strategy for the targeted analysis of protein and peptide metabolites

The detection and quantitation of exogenously administered biological macromolecules (e.g. vaccines, peptide and protein therapeutics) and their metabolites is frequently complicated by the presence of a complex endogenous mixture of closely related compounds. We describe a method that incorporates stable isotope labeling of the compound of interest allowing the selective screening of the intact molecule and all metabolites using a modified precursor ion scan. This method involves monitoring the low molecular weight fragment ions produced during MS/MS that distinguish isotopically labelled material from related endogenous compounds. All isotopically labelled substances can be selected using this scanning technique for further analysis whilst other unlabelled and irrelevant substances are ignored. The potential for this technique to be used in metabolism and pharmacokinetic experiments is discussed with specific examples looking at the metabolism of α-synuclein in serum and the brain

Expression of the wilms' tumor gene WT1 in human malignant mesothelioma cell lines and relationship to platelet‐derived growth factor A and insulin‐like growth factor 2 expression

Mutations in the WT1 tumor suppressor gene are known to contribute to the development of Wilms' tumor (WT) and associated gonadal abnormalities. WT1 is expressed principally in the fetal kidney, developing gonads, and spleen and also in the mesothelium, which lines the coelomic cavities. These tissues develop from mesenchymal components that have subsequently become epithelialized, and it has therefore been proposed that WT1 may play a role in this transition of cell types. To test the possible involvement of this gene in malignant mesothelioma, we have first studied its expression in a panel of human normal and malignant mesothelial cell lines. WT1 mRNA expression levels varied greatly between the cell lines and no specific chromosomal aberration on 11p, which could be related to the variation in WT1 expression in these cell lines, was observed. Furthermore, no gross deletions, rearrangements, or functionally inactivating point mutations in the WT1 coding region were identified. All four WT1 splice variants were observed at similar levels in these cell lines. The WT1 gene encodes a zinc‐finger transcription factor and the four protein isoforms are each believed to act as transcriptional repressors of certain growth factor genes. Lack of WT1 expression is thus predicted to result in growth stimulation of tumor cells. Binding of one particular WT1 isoform construct to the insulin‐like growth factor 2 (IGF2) and platelet‐derived growth factor A (PDGFA) gene promoters has been demonstrated to result in repression of these genes in transient transfection studies. Analysis of IGF2 and PDGFA mRNA expression levels compared with WT1 mRNA expression levels failed to demonstrate an inverse correlation in the mesothelial cell lines, which endogenously express these genes. Finally, the putative role of WT1 in the transition of cell types was investigated. No obvious correlation between WT1 expression levels and cell morphology of the malignant mesothelial cell lines was evident from this study. Moreover, no change in WT1 expression was observed in normal mesothelial cells which were, by alteration of culture conditions, manipulated to switch from the mesenchymal to epithelial morphology.</p

Expression of the wilms' tumor gene WT1 in human malignant mesothelioma cell lines and relationship to platelet‐derived growth factor A and insulin‐like growth factor 2 expression

Mutations in the WT1 tumor suppressor gene are known to contribute to the development of Wilms' tumor (WT) and associated gonadal abnormalities. WT1 is expressed principally in the fetal kidney, developing gonads, and spleen and also in the mesothelium, which lines the coelomic cavities. These tissues develop from mesenchymal components that have subsequently become epithelialized, and it has therefore been proposed that WT1 may play a role in this transition of cell types. To test the possible involvement of this gene in malignant mesothelioma, we have first studied its expression in a panel of human normal and malignant mesothelial cell lines. WT1 mRNA expression levels varied greatly between the cell lines and no specific chromosomal aberration on 11p, which could be related to the variation in WT1 expression in these cell lines, was observed. Furthermore, no gross deletions, rearrangements, or functionally inactivating point mutations in the WT1 coding region were identified. All four WT1 splice variants were observed at similar levels in these cell lines. The WT1 gene encodes a zinc‐finger transcription factor and the four protein isoforms are each believed to act as transcriptional repressors of certain growth factor genes. Lack of WT1 expression is thus predicted to result in growth stimulation of tumor cells. Binding of one particular WT1 isoform construct to the insulin‐like growth factor 2 (IGF2) and platelet‐derived growth factor A (PDGFA) gene promoters has been demonstrated to result in repression of these genes in transient transfection studies. Analysis of IGF2 and PDGFA mRNA expression levels compared with WT1 mRNA expression levels failed to demonstrate an inverse correlation in the mesothelial cell lines, which endogenously express these genes. Finally, the putative role of WT1 in the transition of cell types was investigated. No obvious correlation between WT1 expression levels and cell morphology of the malignant mesothelial cell lines was evident from this study. Moreover, no change in WT1 expression was observed in normal mesothelial cells which were, by alteration of culture conditions, manipulated to switch from the mesenchymal to epithelial morphology.</p

Quantitative lipidomic analysis of Ascaris suum

Ascaris is a soil-transmitted nematode that causes ascariasis, a neglected tropical disease affecting predominantly children and adolescents in the tropics and subtropics. Approximately 0.8 billion people are affected worldwide, equating to 0.86 million disability-adjusted life-years (DALYs). Exploring the molecular biology of Ascaris is important to gain a better understanding of the host-parasite interactions and disease processes, and supports the development of novel interventions. Although advances have been made in the genomics, transcriptomics and proteomics of Ascaris, its lipidome has received very limited attention. Lipidomics is an important sub-discipline of systems biology, focused on exploring lipids profiles in tissues and cells, and elucidating their biological and metabolic roles. Here, we characterised the lipidomes of key developmental stages and organ systems of Ascaris of porcine origin via high throughput LC-MS/MS. In total, > 500 lipid species belonging to 18 lipid classes within three lipid categories were identified and quantified-in precise molar amounts in relation to the dry weight of worm material-in different developmental stages/sexes and organ systems. The results showed substantial differences in the composition and abundance of lipids with key roles in cellular processes and functions (e.g. energy storage regulation and membrane structure) among distinct stages and among organ systems, likely reflecting differing demands for lipids, depending on stage of growth and development as well as the need to adapt to constantly changing environments within and outside of the host animal. This work provides the first step toward understanding the biology of lipids in Ascaris, with possibilities to work toward designing new interventions against ascariasis. Author summary Lipids are of vital importance in the biology of parasitic worms, particularly in relation to cellular membranes, energy storage, and intra- and intercellular signalling. However, very little is known about the biology of lipids in parasitic nematodes. Using a high-throughput LC-MS/MS approach, we characterised the first global lipidome for Ascaris. We investigated the lipid composition and abundance in key developmental stages/sexes as well as the organ systems of Ascaris. We observed substantial differences in lipid composition and abundance among these stages/sexes and among the organ systems studied. The findings provide a basis to start to understand lipid biology in Ascaris, with possible implications for developing new interventions against ascariasis

Do primate action plans work?

John Oates authored the first primate conservation Action Plan in 1986, which assessed the status of and proposed conservation actions for all mainland African primate species. A revised version of the continent-wide plan was published in 1996, but since then, action plans have generally evolved into prioritizing actions for specific species, often within defined landscapes. We will review and evaluate the content and success of conservation action plans for the nine currently recognized taxa of chimpanzees and gorillas in Africa. Since 2003, six detailed action plans and one population viability analysis have been published, covering priority actions and landscapes for seven of the nine great ape taxa in Africa. Two further action plans (for gorillas and chimpanzees in Eastern DRC and for bonobos) are in the final stages of review and may also be included in the analysis. Assessments for western chimpanzees, Cross River gorillas, western lowland gorillas and central chimpanzees have been peer reviewed, and we will consider their recommendations and the challenges of quantitatively evaluating the success of primate conservation action plans

Detecting the Companions and Ellipsoidal Variations of RS CVn Primaries: II. omicron Draconis, a Candidate for Recent Low-Mass Companion Ingestion

To measure the stellar and orbital properties of the metal-poor RS CVn binary o Draconis (o Dra), we directly detect the companion using interferometric observations obtained with the Michigan InfraRed Combiner at Georgia State University's Center for High Angular Resolution Astronomy (CHARA) Array. The H-band flux ratio between the primary and secondary stars is the highest confirmed flux ratio (370 +/- 40) observed with long-baseline optical interferometry. These detections are combined with radial velocity data of both the primary and secondary stars, including new data obtained with the Tillinghast Reflector Echelle Spectrograph on the Tillinghast Reflector at the Fred Lawrence Whipple Observatory and the 2-m Tennessee State University Automated Spectroscopic Telescope at Fairborn Observatory. We determine an orbit from which we find model-independent masses and ages of the components (M_A = 1.35 +\- 0.05 M_Sun, M_B = 0.99 +\- 0.02 M_Sun, system age = 3.0 -\+ 0.5 Gyr). An average of a 23-year light curve of o Dra from the Tennessee State University Automated Photometric Telescope folded over the orbital period newly reveals eclipses and the quasi-sinusoidal signature of ellipsoidal variations. The modeled light curve for our system's stellar and orbital parameters confirm these ellipsoidal variations due to the primary star partially filling its Roche lobe potential, suggesting most of the photometric variations are not due to stellar activity (starspots). Measuring gravity darkening from the average light curve gives a best-fit of beta = 0.07 +\- 0.03, a value consistent with conventional theory for convective envelope stars. The primary star also exhibits an anomalously short rotation period, which, when taken with other system parameters, suggests the star likely engulfed a low-mass companion that had recently spun-up the star.Comment: 14 pages, 13 figures, Accepted to Ap

Extended H? emission line sources from UWISH2

We present the extended source catalogue for the UKIRT Wide Field Infrared Survey for H2 (UWISH2). The survey is unbiased along the inner Galactic Plane from l ? 357° to l ? 65° and |b| ? 1.5° and covers 209 deg2. A further 42.0 and 35.5 deg2 of high dust column density regions have been targeted in Cygnus and Auriga. We have identified 33 200 individual extended H2 features. They have been classified to be associated with about 700 groups of jets and outflows, 284 individual (candidate) planetary nebulae, 30 supernova remnants and about 1300 photodissociation regions. We find a clear decline of star formation activity (traced by H2 emission from jets and photodissociation regions) with increasing distance from the Galactic Centre. About 60 per cent of the detected candidate planetary nebulae have no known counterpart and 25 per cent of all supernova remnants have detectable H2 emission associated with them

Multiwavelength Variations of 3C 454.3 during the November 2010 to January 2011 Outburst

We present multiwavelength data of the blazar 3C 454.3 obtained during an extremely bright outburst from November 2010 through January 2011. These include flux density measurements with the Herschel Space Observatory at five submillimeter-wave and far-infrared bands, the Fermi Large Area Telescope at gamma-ray energies, Swift at X-ray, ultraviolet (UV), and optical frequencies, and the Submillimeter Array at 1.3 mm. From this dataset, we form a series of 52 spectral energy distributions (SEDs) spanning nearly two months that are unprecedented in time coverage and breadth of frequency. Discrete correlation anlaysis of the millimeter, far-infrared, and gamma-ray light curves show that the variations were essentially simultaneous, indicative of co-spatiality of the emission, at these wavebands. In contrast, differences in short-term fluctuations at various wavelengths imply the presence of inhomegeneities in physical conditions across the source. We locate the site of the outburst in the parsec-scale core, whose flux density as measured on 7 mm Very Long Baseline Array images increased by 70 percent during the first five weeks of the outburst. Based on these considerations and guided by the SEDs, we propose a model in which turbulent plasma crosses a conical standing shock in the parsec-scale region of the jet. Here, the high-energy emission in the model is produced by inverse Compton scattering of seed photons supplied by either nonthermal radiation from a Mach disk, thermal emission from hot dust, or (for X-rays) synchrotron radiation from plasma that crosses the standing shock. For the two dates on which we fitted the model SED to the data, the model corresponds very well to the observations at all bands except at X-ray energies, where the spectrum is flatter than observed.Comment: Accepted for publication in Astrophysical Journal. 82 pages, 13 figure

Regional Action Plan for the Conservation of the Cross River Gorilla (Gorilla gorilla diehli)

From Executive Summary: This document represents the consensus of experts who met at a workshop in April 2006 in Calabar, Cross River State, Nigeria, to formulate a set of priority actions that would increase the survival prospects for the Cross River gorilla (Gorilla gorilla diehli). The Cross River gorilla is recognized by IUCN as Critically Endangered, and is the most threatened taxon of ape in Africa. It is the most westerly and northerly form of gorilla, and occurs only in a limited area around the mountainous headwaters of the Cross River, straddling the border between Cameroon and Nigeria. Participants at the 2006 workshop, which built upon the outcomes of previous meetings in Calabar in 2001 and Limbe, Cameroon, in 2003, included representatives of forestry and wildlife conservation agencies from the two range countries, of local and international nongovernmental conservation and development organizations, and of university-based researchers

The insulin A-chain epitope recognized by human T cells is posttranslationally modified

The autoimmune process that destroys the insulin-producing pancreatic β cells in type 1 diabetes (T1D) is targeted at insulin and its precursor, proinsulin. T cells that recognize the proximal A-chain of human insulin were identified recently in the pancreatic lymph nodes of subjects who had T1D. To investigate the specificity of proinsulin-specific T cells in T1D, we isolated human CD4+ T cell clones to proinsulin from the blood of a donor who had T1D. The clones recognized a naturally processed, HLA DR4–restricted epitope within the first 13 amino acids of the A-chain (A1–13) of human insulin. T cell recognition was dependent on the formation of a vicinal disulfide bond between adjacent cysteine residues at A6 and A7, which did not alter binding of the peptide to HLA DR4. CD4+ T cell clones that recognized this epitope were isolated from an HLA DR4+ child with autoantibodies to insulin, and therefore, at risk for T1D, but not from two healthy HLA DR4+ donors. We define for the first time a novel posttranslational modification that is required for T cell recognition of the insulin A-chain in T1D