Contamination of depressional wetlands in the Mpumalanga Lake District of South Africa near a global emission hotspot.

The Mpumalanga Lake District (MLD) of South Africa hosts a regionally unique cluster of water bodies of great importance for wetland biodiversity. It is also located close to a global hotspot for coal-fired power station emissions but the local impacts from these sources of pollution are poorly understood. Sediment cores from three contrasting wetlands were 210Pb dated and analysed for a range of contaminants linked to fossil fuel combustion, including trace elements, Hg, sulphur and spheroidal carbonaceous fly-ash particles (SCPs). At the two sites with pre-industrial (1900) baseline sediments, Pb, Zn and especially Cr concentrations and fluxes showed significant increases in the impact period (post-1975). Mercury showed the greatest proportional increase in flux (>4-fold) of all trace metals. Mercury and sulphur concentrations and fluxes showed highly significant correlations with emissions over the corresponding periods, while SCPs in sediments also closely tracked emissions. In a global context, levels of sediment contamination are relatively minor compared with other heavily industrialised regions, with only Cr exceeding the sediment Probable Effects Concentration for biological impact post-1975. Despite the relatively large increases in Hg, concentrations do not reach the Threshold Effects Concentration. The unexpectedly low levels of contamination may be due to i) low levels of many trace contaminants in South African coals compared to global averages, ii) prevailing recirculation patterns which transport pollution away from the study area during the wet season, minimising wet deposition, and iii) pollutant remobilisation through desiccation of wetlands or volatilization. The effects of hydrology and sediment accumulation rates lead to differential transport and preservation of organic-associated and more volatile contaminants (e.g. Hg, S) relative to non-volatile trace elements in wetlands of the MLD. The greatest fluxes of Hg and S are recorded in the site with the highest catchment: lake area ratio, lowest salinity and greatest sediment organic matter content.

Trace element contaminants associated with historic gold mining in sediments of dams and pans across Benoni, South Africa.

As a historic gold mining area, the City of Benoni has numerous water bodies close to major tailings dams and mine dumps. Here we assess spatial patterns in the sediment geochemistry of five dams and four natural pans within a 5-km radius of the core mining area to determine the degree of contamination7 by mining-associated pollutants. XRF analysis was used with a geoaccumulation index to assess the degree of contamination above background levels. Prevailing winds blow from the north and north-west with less dominant winds from the east. Sediment concentrations of As, Cu, Pb, Ni and Zn are highly correlated across the region, suggestive of a common source. Except for one pan showing evidence of local industrial pollution, the most contaminated sites are the dams to the west of the mine dumps where concentrations of As, Cu, Pb and Zn increase towards the central mining area, with highest trace metal concentrations in Kleinfontein Dam, adjacent to a reworked mine dump. Sites upwind of the central mining area showed little evidence of mining-related contamination. Trace contaminant concentrations in sediments of these dams are much greater than those found in the nearby Springs-Blesbokspruit wetlands adjacent to more recently active mines. The potential risks to aquatic ecosystems and recreational users of these urban and suburban water bodies from these wind-blown, legacy mining contaminants merit further investigation.

Multi-polygenic score approach to trait prediction.

A primary goal of polygenic scores, which aggregate the effects of thousands of trait-associated DNA variants discovered in genome-wide association studies (GWASs), is to estimate individual-specific genetic propensities and predict outcomes. This is typically achieved using a single polygenic score, but here we use a multi-polygenic score (MPS) approach to increase predictive power by exploiting the joint power of multiple discovery GWASs, without assumptions about the relationships among predictors. We used summary statistics of 81 well-powered GWASs of cognitive, medical and anthropometric traits to predict three core developmental outcomes in our independent target sample: educational achievement, body mass index (BMI) and general cognitive ability. We used regularized regression with repeated cross-validation to select from and estimate contributions of 81 polygenic scores in a UK representative sample of 6710 unrelated adolescents. The MPS approach predicted 10.9% variance in educational achievement, 4.8% in general cognitive ability and 5.4% in BMI in an independent test set, predicting 1.1%, 1.1%, and 1.6% more variance than the best single-score predictions. As other relevant GWA analyses are reported, they can be incorporated in MPS models to maximize phenotype prediction. The MPS approach should be useful in research with modest sample sizes to investigate developmental, multivariate and gene-environment interplay issues and, eventually, in clinical settings to predict and prevent problems using personalized interventions.

Sinks for nitrogen inputs in terrestrial ecosystems: a meta-analysis of 15N tracer field studies.

Effects of anthropogenic nitrogen (N) deposition and the ability of terrestrial ecosystems to store carbon (C) depend in part on the amount of N retained in the system and its partitioning among plant and soil pools. We conducted a meta-analysis of studies at 48 sites across four continents that used enriched 15N isotope tracers in order to synthesize information about total ecosystem N retention (i.e., total ecosystem 15N recovery in plant and soil pools) across natural systems and N partitioning among ecosystem pools. The greatest recoveries of ecosystem 15N tracer occurred in shrublands (mean, 89.5%) and wetlands (84.8%) followed by forests (74.9%) and grasslands (51.8%). In the short term (< 1 week after 15N tracer application), total ecosystem 15N recovery was negatively correlated with fine-root and soil 15N natural abundance, and organic soil C and N concentration but was positively correlated with mean annual temperature and mineral soil C:N. In the longer term (3-18 months after 15N tracer application), total ecosystem 15N retention was negatively correlated with foliar natural-abundance 15N but was positively correlated with mineral soil C and N concentration and C:N, showing that plant and soil natural-abundance 15N and soil C:N are good indicators of total ecosystem N retention. Foliar N concentration was not significantly related to ecosystem 15N tracer recovery, suggesting that plant N status is not a good predictor of total ecosystem N retention. Because the largest ecosystem sinks for 15N tracer were below ground in forests, shrublands, and grasslands, we conclude that growth enhancement and potential for increased C storage in aboveground biomass from atmospheric N deposition is likely to be modest in these ecosystems. Total ecosystem 15N recovery decreased with N fertilization, with an apparent threshold fertilization rate of 46 kg N x ha(-1) x yr(-1) above which most ecosystems showed net losses of applied 15N tracer in response to N fertilizer addition.

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds.

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl](1/2) at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys(36)-Cys(49) and two disulfide bonds formed by two pair of consecutive cysteines, Cys(22)-Cys(23) and Cys(56)-Cys(57), a unique disulfide structure of polypeptide that has not been documented previously.

Pathway of oxidative folding of bovine alpha-interferon: predominance of native disulfide-bonded folding intermediates.

Bovine alpha-interferon (BoINF-alpha) is a single polypeptide protein containing 166 amino acids, two disulfide bonds (Cys1-Cys99 and Cys29-Cys138), and five stretches of alpha-helical structure. The pathway of oxidative folding of BoINF-alpha has been investigated here. Of the eight possible one- and two-disulfide isomers, only two nativelike one-disulfide isomers, BoINF-alpha (Cys1-Cys99) and BoINF-alpha (Cys29-Cys138), predominate as intermediates along the folding pathway. More strikingly, alpha-helical structures formed almost quantitatively before any detectable formation of a disulfide bond. This is demonstrated by the observation that fully reduced BoINF-alpha (starting material of oxidative folding) and reduced carboxymethylated BoINF-alpha both exhibit alpha-helical structure content indistinguishable form that of native BoINF-alpha. The folding mechanism of BoINF-alpha appears to be compatible with the framework model, in which secondary structures fold first, followed by docking (compaction) of preformed secondary structural elements yielding the native structure.

Conformational stability of secretory leucocyte protease inhibitor: a protein with no hydrophobic core and very little secondary structure.

Conformational stability of proteins (including disulfide containing proteins) has been routinely characterized by spectroscopic techniques. Proteins which lack adequate signal of circular dichroism may require unconventional technique. Secretory Leucocyte Protease Inhibitor (SLPI) is a 107 amino acids protein with a high density of disulfide pairing (eight). The native SLPI has no hydrophobic core and contains very little hydrogen bonded secondary structure [Gruetter, M., Fendrich, G., Huber, R., and Bode, W. (1988) The 2.5 A X-ray crystal structure of the acid stable proteinase inhibitor from human mucous secretions analyzed in its complex with bovine alpha-chymotrypsin. The EMBO J. 7, 345-352.]. In this study, conformational stability of SLPI has been investigated by the method of disulfide scrambling, which permits quantification of the native and denatured (scrambled) proteins by HPLC. Due to high heterogeneity of denatured SLPI, the native and scrambled SLPI are extensively overlapped on HPLC. This impediment was further overcome by the development of a novel method which distinguishes the native and scrambled isomers of SLPI by exploiting the relative stability of their disulfide bonds. The study reveals mid-point denaturation of SLPI at 1.36 M of GdmSCN, 4.0 M of GdmCl and >8 M urea. Based on the GdmCl denaturation curve, the unfolding free energy (DeltaG(H20)) of SLPI was estimated to be 4.56 kcal/mol. The results of our studies suggest an alternative strategy for analyzing conformational stability of disulfide proteins that are not suitable to the conventional spectroscopic techniques.

Pathway of oxidative folding of secretory leucocyte protease inhibitor: an 8-disulfide protein exhibits a unique mechanism of folding.

Secretory leucocyte protease inhibitor (SLPI) is a 107-amino acid protein with a high density of disulfide pairing (eight). The mechanism of oxidative folding of reduced and denatured SLPI has been investigated here. Despite an exceedingly large number of possible folding intermediates ( approximately 46 million disulfide isomers) and their potential to complicate the refolding process, oxidative folding of SLPI turns out to be surprisingly simple and efficient. Complete oxidative folding and a near-quantitative recovery of the native SLPI can be achieved in a simple buffer solution using air oxidation without any supplementing thiol catalyst or redox agent, a phenomenon that has not yet been observed with other disulfide proteins. Because of the heterogeneity and extensive overlapping of folding intermediates, identification of the predominant intermediate was unfeasible. Nonetheless, studies of reductive unfolding of native SLPI and oxidative folding of a six-disulfide variant of SLPI enable us to propose an underlying mechanism accounting for the unique folding efficiency of SLPI in the absence of a redox agent. Our studies indicate that oxidative folding of SLPI undergoes heterogeneous populations of one-, two-, three-, four-, five-, six-, and seven-disulfide isomers, including two nativelike isomers, SLPI-6A and SLPI-7A, as transient intermediates. Formation of the last two native disulfide bonds leading to the conversion of SLPI-6A --> SLPI-7A --> N-SLPI is relatively slow and represents the final stage of oxidative folding. Most importantly, free cysteines of SLPI-6A and SLPI-7A also act as a thiol catalyst in promoting the disulfide shuffling of diverse non-native intermediates accumulated along the folding pathway. This explains why a near-quantitative recovery of N-SLPI can be achieved in the absence of any thiol catalyst and redox agent. Properties of SLPI-6A and SLPI-7A were investigated and compared to those of other documented kinetic intermediates of oxidative folding. The correlation between the mechanism of SLPI folding and the three-dimensional structure of SLPI is also elaborated.

Fully oxidized scrambled isomers are essential and predominant folding intermediates of cardiotoxin-III.

Scrambled isomers (X-isomers) are fully oxidized, non-native isomers of disulfide proteins. They have been shown to represent important intermediates along the pathway of oxidative folding of numerous disulfide proteins. A simple method to assess whether X-isomers present as folding intermediate is to conduct oxidative folding of fully reduced protein in the alkaline buffer alone without any supplementing thiol catalyst or redox agent. Cardiotoxin-III (CTX-III) contains 60 amino acids and four disulfide bonds. The mechanism of oxidative folding of CTX-III has been systematically characterized here by analysis of the acid trapped folding intermediates. Folding of CTX-III was shown to proceed sequentially through 1-disulfide, 2-disulfide, 3-disulfide and 4-disulfide (scrambled) isomers as folding intermediates to reach the native structure. When folding of CTX-III was performed in the buffer alone, more than 97% of the protein was trapped as 4-disulfide X-isomers, unable to convert to the native structure due to the absence of thiol catalyst. In the presence of thiol catalyst (GSH) or redox agents (GSH/GSSG), the recovery of native CTX-III was 80-85%. These results demonstrate that X-isomers play an essential and predominant role in the oxidative folding of CTX-III.

Nitrate leaching as a confounding factor in chemical recovery from acidification in UK upland waters.

Over the period 1988-2002, data from 18 of the 22 lakes and streams in the UK Acid Waters Monitoring Network (AWMN) show clear trends of declining excess sulphate concentrations in response to reductions in sulphur deposition, but fewer trends in increasing pH or alkalinity. There has been no significant decline in the deposition of total nitrogen over the same period, and no sites show a trend in nitrate concentration. Peak nitrate concentrations have already surpassed excess sulphate on occasion in half of the AWMN sites. Furthermore, current understanding of terrestrial N saturation processes suggests that nitrate leaching from soils may increase, even under a constant N deposition load. Best-case projections indicate that nitrate will overtake sulphate as the major excess acid anion in many sites within 10 years, while worst-case predictions with steady-state models suggest that in the longer-term, nitrate could become the dominant excess acid anion in most of the UK.

Hydrogen-transfer reactions which generate new imine, imido, and trimethylenemethane complexes of tantalum.

The reactions of Cp*TaMe3C1 (Cp* = qs-CsMeS) with a variety of alkali-metal alkoxide, alkylamide, and alkyl reagents have been examined. Reaction with LiNMe2 produces Cp*Ta(NMez)Me3, which decomposes at 25 OC to an imine (or metallaazirane) complex, Cp*Ta(CH2NMe)Me2. The decomposition is a first-order, unimolecular process with a large kinetic isotope effect (kH/kD = 9.7). Monoalkylamides (LiNHR) react with Cp*TaMe3C1 to form imido complexes Cp*Ta(NR)Mez. Reaction of Cp*TaMe3C1 with lithium diisopropylamide forms a bridging methylene complex, Cp*Me2Ta(pCH2)(p-H)zTaMezCp*. The alkoxide compounds Cp*Ta(OR)Me3 (R = Me, CHMe2, CMe3) are very stable and decompose only over 100 OC. Alkyl complexes are stable only if the alkyl group does not have j3-hydrogens. Treatment of Cp*TaMe3C1 with (2-methylally1)magneium bromide affords an unstable tantalum 2-methylallyl compound, which decomposes cleanly to the trimethylenemethanec omplex Cp*TaMeZ(q4-C(CH2),)T. he rates of hydrogen abstraction or elimination processes in this system correlate with the nature of the atom bound to tantalum: for reactions involving a /3-hydrogen transfer the order is C > N > 0, while the facility of a-hydrogen abstraction reactions appear to decrease in the reverse order N > C. These reactivity patterns appear to reflect the variance in T a x , Ta-N, and Ta-O bond energies in this series. Hydrogenation of the imido compounds (Cp*Ta(NR)Me2) in the presence of phosphine ligands yields new examples of imido hydride complexes Cp*Ta(NR)H,(L) (L = PMe3, PMe2(C6Hs); R = CMe3, CHzCMe3). A moderately stable alkyl hydride complex, Cp*Ta(CH2NMe)Me(PMe3)H, has also been prepared.