Collaborative learning exercises for Teaching Protein Mass Spectrometry [post-print]

A collaborative learning module for teaching protein mass spectrometry has been developed to overcome common obstacles to incorporating the modern topic of biological mass spectrometry into the undergraduate chemistry curriculum. Protein mass spectrometry data is provided to eliminate the need for expensive instrumentation, and an instructor’s manual gives necessary details for those unfamiliar with the topic. The first section provides background information on proteins and the field of proteomics. The second section describes the use of electrospray ionization to determine the molecular weight of a protein. The third section shows how to identify a protein using peptide mass mapping, and the fourth section describes tandem MS experiments for de novo peptide sequencing. Each section also includes lessons on the analytical instrumentation used to make mass measurements including electrospray ionization, matrix assisted laser desorption ionization, and time-of-flight mass spectrometry. The module includes preclass reading assignments and small group problem solving exercises to be used during class sessions. The module was implemented over several semesters at both a small liberal arts college and a large research university. Assessment data from both institutions suggest that the module is effective in helping students to learn about mass-spectrometry-based proteomics. This freely available resource will assist instructors in introducing these topics to the undergraduate curriculum

Novel approaches to investigating spatial variability in channel bank total phosphorus at the catchment scale

Phosphorus (P) is often a limiting nutrient that leads to the eutrophication of aquatic systems. While dissolved P forms are the most bioavailable, the form, mobility, transport and fate of P are directly related to its association with fine-grained riverine sediment. Therefore, to implement successful P catchment management strategies it is important to understand the relative contribution of different sediment sources to P loads across the river continuum. While agricultural topsoil and, to a lesser extent, riverbed sediment are important sources of sediment-associated P, channel banks have been shown to be an important sediment source in some catchments. However, comparatively little is known about the P concentration and corresponding spatial variability in channel bank sediment and the associated implications for catchment management. The present study examines the spatial variability of P associated with channel bank profiles within a series of three nested catchments using both non-spatial and spatial statistical methods, where for the latter, a novel spatial approach was used to estimate the spatial averages and variances of P in channel bank sediment along the stream network. Channel bank P concentrations were compared to factors such as catchment scale, stream order, land use, bank exposure and location along the stream network. Concentrations of TP ranged between 129.6 and 1206.9 mg P kg⁻¹ of which the water extractable P (WEP) content ranged from 0.01 to 0.12%. Stream order was found to influence TP concentrations, while land use and catchment scale provided only a moderate influence. This suggested that focussing channel bank sampling strategies at the largest catchment scale would capture key drivers of TP variability provided stream order is sufficiently represented. Whether the bank was had limited vegetation and was exposed and potentially eroding had a slight influence on TP variability in second-order stream banks in the larger of the two nested catchments. However, the slightly lower TP concentrations measured at these sites indicated that banks that are actually eroding may be contributing less TP than the total channel bank TP values measured across the catchments as a whole. The results of an explicitly spatial analysis demonstrated that local channel bank TP averages and TP variances vary along the stream network. Specifically, the most accurate spatial predictor of TP was local TP means with the use of ‘crow flies’ rather than stream network distances. Local TP variances were used to provide optimal designs for future channel bank TP sampling campaigns, given available resources. Throughout, both standard and outlier-resistant statistical analyses were applied to improve interpretation of the study findings

Current advisory interventions for grazing ruminant farming cannot close exceedance of modern background sediment loss – Assessment using an instrumented farm platform and modelled scaling out

Water quality impairment by elevated sediment loss is a pervasive problem for global water resources. Sediment management targets identify exceedance or the sediment loss ‘gap’ requiring mitigation. In the UK, palaeo-limnological reconstruction of sediment loss during the 100–150 years pre-dating the post-World War II intensification of agriculture, has identified management targets (0.20−0.35 t ha−1 yr−1) representing ‘modern background sediment delivery to rivers’. To assess exceedance on land for grazing ruminant farming, an integrated approach combined new mechanistic evidence from a heavily-instrumented experimental farm platform and a scaling out framework of modelled commercial grazing ruminant farms in similar environmental settings. Monitoring (2012–2016) on the instrumented farm platform returned sediment loss ranges of 0.11−0.14 t ha−1 yr−1 and 0.21−0.25 t ha−1 yr-1 on permanent pasture, compared with between 0.19−0.23 t ha−1 yr-1 and 0.43−0.50 t ha−1 yr−1and 0.10−0.13 t ha−1 yr−1and 0.25−0.30 t ha−1 yr-1 on pasture with scheduled plough and reseeds. Excess sediment loss existed on all three farm platform treatments but was more extensive on the two treatments with scheduled plough and reseeds. Excessive sediment loss from land used by grazing ruminant farming more strategically across England, was estimated to be up to >0.2 t ha−1 yr−1. Modelled scenarios of alternative farming futures, based on either increased uptake of interventions typically recommended by visual farm audits, or interventions selected using new mechanistic understanding for sediment loss from the instrumented farm platform, returned minimum sediment loss reductions. On the farm platform these were 2.1 % (up to 0.007 t ha−1 yr−1) and 5.1 % (up to 0.018 t ha−1 yr-1). More strategically, these were up to 2.8 % (0.014 t ha−1 yr−1) and 4.1 % (0.023 t ha−1 yr−1). Conventional on-farm measures will therefore not fully mitigate the sediment loss gap, meaning that more severe land cover change is required

Nitric Oxide Is an Upstream Signal of Vascular Endothelial Growth Factor-induced Extracellular Signal-regulated Kinase½ Activation in Postcapillary Endothelium

Abstract We recently demonstrated that nitric oxide (NO) significantly contributes to the mitogenic effect of vascular endothelial growth factor (VEGF), suggesting a role for the NO pathway in the signaling cascade following kinase-derivative receptor activation in vascular endothelium. The aim of this study was to investigate the intracellular pathways linked to VEGF/NO-induced endothelial cell proliferation. We assessed the activity of the mitogen-activated protein kinase (MAPK) that is specifically activated by growth factors, extracellular-regulated kinase (ERK½), on cultured microvascular endothelium isolated from coronary postcapillary venules. ERK½ was immunoprecipitated, and its activity was assessed with an immunocomplex kinase assay. In endothelial cells exposed for 5 min to the NO donor drug sodium nitroprusside at a concentration of 100 μm, ERK½ activity significantly increased. VEGF produced a time- and concentration-dependent activation of ERK½. Maximal activity was obtained after 5 min of stimulation at a concentration of 10 ng/ml. The specific MAPK kinase inhibitor PD 98059 abolished ERK½ activation and endothelial cell proliferation in a concentration-dependent manner in response to VEGF and sodium nitroprusside. The NO synthase inhibitorN ω-monomethyl-l-arginine, as well as the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, blocked the activation of ERK½ induced by VEGF, suggesting that NO and cGMP contributed to the VEGF-dependent ERK½ activation. These results demonstrate for the first time that kinase-derivative receptor activation triggers the NO synthase/guanylate cyclase pathway to activate the MAPK cascade and substantiates the hypothesis that the activation of ERK½ is necessary for VEGF-induced endothelial cell proliferation

Tourism income and economic growth in Greece: Empirical evidence from their cyclical components

This paper examines the relationship between the cyclical components of Greek GDP and international tourism income for Greece for the period 1976–2004. Using spectral analysis the authors find that cyclical fluctuations of GDP have a length of about nine years and that international tourism income has a cycle of about seven years. The volatility of tourism income is more than eight times the volatility of the Greek GDP cycle. VAR analysis shows that the cyclical component of tourism income is significantly influencing the cyclical component of GDP in Greece. The findings support the tourism-led economic growth hypothesis and are of particular interest and importance to policy makers, financial analysts and investors dealing with the Greek tourism industry

Phosphate stable oxygen isotope variability within a temperate agricultural soil

In this study, we conduct a spatial analysis of soil total phosphorus (TP), acid extractable phosphate (PO4) and the stable oxygen (O) isotope ratio within the PO4 molecule (δ18OPO4) from an intensively managed agricultural grassland site. Total P in the soil was found to range from 736 to 1952 mg P kg− 1, of which between 12 and 48% was extractable using a 1 M HCl (HClPO4) solution with the two variables exhibiting a strong positive correlation. The δ18OPO4 of the extracted PO4 ranged from 17.0 to 21.6‰ with a mean of 18.8‰ (± 0.8). While the spatial variability of Total P has been researched at various scales, this is the first study to assess the variability of soil δ18OPO4 at a field-scale resolution. We investigate whether or not δ18OPO4 variability has any significant relationship with: (i) itself with respect to spatial autocorrelation effects; and (ii) HClPO4, elevation and slope - both globally and locally. Results indicate that δ18OPO4 was not spatially autocorrelated; and that δ18OPO4 was only weakly related to HClPO4, elevation and slope, when considering the study field as a whole. Interestingly, the latter relationships appear to vary in strength locally. In particular, the δ18OPO4 to HClPO4 relationship may depend on the underlying soil class and/or on different field managements that had operated across an historical north-south field division of the study field, a division that had been removed four years prior to this study

A Genome-Wide Collection of Mos1 Transposon Insertion Mutants for the C. elegans Research Community

Methods that use homologous recombination to engineer the genome of C. elegans commonly use strains carrying specific insertions of the heterologous transposon Mos1. A large collection of known Mos1 insertion alleles would therefore be of general interest to the C. elegans research community. We describe here the optimization of a semi-automated methodology for the construction of a substantial collection of Mos1 insertion mutant strains. At peak production, more than 5,000 strains were generated per month. These strains were then subject to molecular analysis, and more than 13,300 Mos1 insertions characterized. In addition to targeting directly more than 4,700 genes, these alleles represent the potential starting point for the engineered deletion of essentially all C. elegans genes and the modification of more than 40% of them. This collection of mutants, generated under the auspices of the European NEMAGENETAG consortium, is publicly available and represents an important research resource

Salivary testosterone levels in preadolescent children

BACKGROUND: Saliva reflects the plasma free fraction of testosterone which is biologically active, and available for uptake by tissues. Testosterone concentration in saliva, though differing slightly from the concentration of unbound testosterone in serum, is in good correlation with the latter, indicating that salivary testosterone provides a reliable method for determination of serum free testosterone. The study aimed to investigate salivary testosterone levels and their changes in preadolescent children and to study sexual dimorphism. METHODS: Testosterone levels were determined in 203 healthy preadolescent children (77 girls and 126 boys) from saliva samples by radioimmunoassay. Sampling was performed once a year with respect to circadian and seasonal fluctuations of testosterone. Data were statistically analyzed by Statgraphic software. RESULTS: Mean salivary testosterone concentrations (± SD) were 0.038 ± 0.012 nmol/L and 0.046 ± 0.026 nmol/L for girls and boys, with the medians 0.035 nmol/L and 0.041 nmol/L, respectively. Statistical analysis did not prove changes in salivary testosterone concentrations in the preadolescent period of life, with an exception of the insignificant fall at the age of 7 years, and an insignificant rise at the age of 9 years in girls. CONCLUSIONS: Generally it can be concluded that salivary testosterone levels in our prepubertal subjects remained stable. There was no significant increase of salivary testosterone levels from the age of 6 until the age of 9 in both sexes. Sexual dimorphism in salivary testosterone levels was proved with significantly higher (p = 0.009) salivary testosterone levels in boys than in girls