Accumulated metal speciation in earthworm populations with multi-generational exposure to metalliferous soils: cell fractionation and high energy synchrotron analyses

Predicting metal bioaccumulation and toxicity in soil organisms is complicated by site-specific biotic and abiotic parameters. In this study we exploited tissue fractionation and digestion techniques, combined with X-ray absorption spectroscopy (XAS), to investigate the whole-body and subcellular distributions, ligand affinities, and coordination chemistry of accumulated Pb and Zn in field populations of the epigeic earthworm Lumbricus rubellus inhabiting three contrasting metalliferous and two unpolluted soils. Our main findings were (i) earthworms were resident in soils with concentrations of Pb and Zn ranging from 1200 to 27 000 mg kg(-1) and 200 to 34 000 mg kg(-1), respectively; (ii) Pb and Zn primarily accumulated in the posterior alimentary canal in nonsoluble subcellular fractions of earthworms; (iii) site-specific differences in the tissue and subcellular partitioning profiles of populations were observed, with earthworms from a calcareous site partitioning proportionally more Pb to their anterior body segments and Zn to the chloragosome-rich subcellular fraction than their acidic-soil inhabiting counterparts; (iv) XAS indicated that the interpopulation differences in metal partitioning between organs were not accompanied by qualitative differences in ligand-binding speciation, because crystalline phosphate-containing pyromorphite was a predominant chemical species in the whole-worm tissues of all mine soil residents. Differences in metal (Pb, Zn) partitioning at both organ and cellular levels displayed by field populations with protracted histories of metal exposures may reflect their innate ecophysiological responses to essential edaphic variables, such as Ca2+ status. These observations are highly significant in the challenging exercise of interpreting holistic biomarker data delivered by "omic" technologies.

Molecular genetic differentiation in earthworms inhabiting a heterogeneous Pb-polluted landscape

A Pb-mine site situated on acidic soil, but comprising of Ca-enriched islands around derelict buildings was used to study the spatial pattern of genetic diversity in Lumbricus rubellus. Two distinct genetic lineages ('A' and 'B'), differentiated at both the mitochondrial (mtDNA COII) and nuclear level (AFLPs) were revealed with a mean inter-lineage mtDNA sequence divergence of approximately 13%, indicative of a cryptic species complex. AFLP analysis indicates that lineage A individuals within one central 'ecological island' site are uniquely clustered, with little genetic overlap with lineage A individuals at the two peripheral sites. FTIR microspectroscopy of Pb-sequestering chloragocytes revealed different phosphate profiles in residents of adjacent acidic and calcareous islands. Bioinformatics found over-representation of Ca pathway genes in ESTPb libraries. Subsequent sequencing of a Ca-transport gene, SERCA, revealed mutations in the protein's cytosolic domain. We recommend the mandatory genotyping of all individuals prior to field-based ecotoxicological assays, particularly those using discriminating genomic technologies.

Enantioselective Intramolecular Michael Addition of Nitronates onto Conjugated Esters: Access to Cyclic γ-Amino Acids with up to Three Stereocenters

A highly stereoselective synthesis of conformationally constrained cyclic γ-amino acids has been devised. The key step involves an intramolecular cyclization of a nitronate onto a conjugated ester, promoted by a bifunctional thiourea catalyst. This methodology has been successfully applied to generate a variety of γ-amino acids, including some containing three contiguous stereocenters, with very high diastereoselectivity and excellent enantioselectivity. It is postulated that an interaction that is key to the success of the process is the simultaneous coordination of the thiourea functionality to both the conjugated ester and the nitronate. Finally, the synthetic utility of these compounds is demonstrated in the synthesis of two dipeptides derived from the C- and N-termini.

Metal bioaccumulation and cellular fractionation in an epigeic earthworm (Lumbricus rubellus): the interactive influences of population exposure histories, site-specific geochemistry and mitochondrial genotype

Subcellular fractionation techniques were used to describe temporal changes (at intervals from T0 to T70 days) in the Pb, Zn and P partitioning profiles of Lumbricus rubellus populations from one calcareous (MDH) and one acidic (MCS) geographically isolated Pb/Zn-mine sites and one reference site (CPF). MDH and MCS individuals were laboratory maintained on their native field soils; CPF worms were exposed to both MDH and MCS soils. Site-specific differences in metal partitioning were found: notably, the putatively metal-adapted populations, MDH and MCS, preferentially partitioned higher proportions of their accumulated tissue metal burdens into insoluble CaPO4-rich organelles compared with naive counterparts, CPF. Thus, it is plausible that efficient metal immobilization is a phenotypic trait characterising metal tolerant ecotypes. Mitochondrial cytochrome oxidase II (COII) genotyping revealed that the populations indigenous to mine and reference soils belong to distinct genetic lineages, differentiated by 13%, with 7 haplotypes within the reference site lineage but fewer (3 and 4, respectively) in the lineage common to the two mine sites. Collectively, these observations raise the possibility that site-related genotype differences could influence the toxico-availability of metals and, thus, represent a potential confounding variable in field-based eco-toxicological assessments.

Iron-regulated surface determinant protein a mediates adhesion of staphylococcus aureus to human corneocyte envelope proteins

The ability of Staphylococcus aureus to colonize the human nares is a crucial prerequisite for disease. IsdA is a major S. aureus surface protein that is expressed during human infection and required for nasal colonization and survival on human skin. In this work, we show that IsdA binds to involucrin, loricrin, and cytokeratin K10, proteins that are present in the cornified envelope of human desquamated epithelial cells. To measure the forces and dynamics of the interaction between IsdA and loricrin (the most abundant protein of the cornified envelope), single-molecule force spectroscopy was used, demonstrating high-specificity binding. IsdA acts as a cellular adhesin to the human ligands, promoting whole-cell binding to immobilized proteins, even in the absence of other S. aureus components (as shown by heterologous expression in Lactococcus lactis). Inhibition experiments revealed the binding of the human ligands to the same IsdA region. This region was mapped to the NEAT domain of IsdA. The NEAT domain also was found to be required for S. aureus whole-cell binding to the ligands as well as to human nasal cells. Thus, IsdA is an important adhesin to human ligands, which predominate in its primary ecological niche.

Asymmetric phase-transfer-catalyzed synthesis of five-membered cyclic gamma-Amino acid precursors

The first example of an intramolecular enantioselective Michael addition of nitronates onto conjugated systems utilizing a chiral phase-transfer catalyst is described. A range of five-membered gamma-nitro esters with up to three stereocentres have been prepared and the relative and absolute configurations proven by chemical and crystallographic methods. The products are rapidly obtained and are precursors to five-membered cyclic gamma-amino acids.

Aldol reaction of butane-2,3-diacetal protected methyl glycerate

The addition of aldehydes to butane-2,3-diacetal has been investigated. The reaction was shown to be both regioselective and diastereoselective by 1H NMR spectroscopy. The configuration of the newly formed stereocenter was determined by Mosher’s ester analysis.

Trapping of palindromic ligands within native transthyretin prevents amyloid formation

Transthyretin (TTR) amyloidosis is a fatal disease for which new therapeutic approaches are urgently needed. We have designed two palindromic ligands, 2,2’-(4,4’-(heptane 1,7-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (mds84) and 2,2’-(4,4’-(undecane-1,11-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (4ajm15), that are rapidly bound by native wild-type TTR in whole serum and even more avidly by amyloidogenic TTR variants. One to one stoichiometry, demonstrable in solution and by MS, was confirmed by X-ray crystallographic analysis showing simultaneous occupation of both T4 binding sites in each tetrameric TTR molecule by the pair of ligand head groups. Ligand binding by native TTR was irreversible under physiological conditions, and it stabilized the tetrameric assembly and inhibited amyloidogenic aggregation more potently than other known ligands. These superstabilizers are orally bioavailable and exhibit low inhibitory activity against cyclooxygenase (COX). They offer a promising platform for development of drugs to treat and prevent TTR amyloidosis.

General aspects of organocatalytic cyclizations

Ring-forming reactions are an essential part of synthetic chemistry and allow access to a range of useful natural products and biologically important molecules. The applications of organocatalysis to the synthesis of functionalized, enantiopure structures really begins where organocatalysis itself begins; with the Hajos-Parrish reaction in the 1970s for the synthesis of steroids using proline. This chapter then will review the uses of organocatalysts in cyclization methodology – from the initial Hajos-Parrish discovery to current advances in the field.

An improved lesion detection approach based on similarity measurement between fuzzy intensity segmentation and spatial probability maps

The application of automatic segmentation methods in lesion detection is desirable. However, such methods are restricted by intensity similarities between lesioned and healthy brain tissue. Using multi-spectral magnetic resonance imaging (MRI) modalities may overcome this problem but it is not always practicable. In this article, a lesion detection approach requiring a single MRI modality is presented, which is an improved method based on a recent publication. This new method assumes that a low similarity should be found in the regions of lesions when the likeness between an intensity based fuzzy segmentation and a location based tissue probabilities is measured. The usage of a normalized similarity measurement enables the current method to fine-tune the threshold for lesion detection, thus maximizing the possibility of reaching high detection accuracy. Importantly, an extra cleaning step is included in the current approach which removes enlarged ventricles from detected lesions. The performance investigation using simulated lesions demonstrated that not only the majority of lesions were well detected but also normal tissues were identified effectively. Tests on images acquired in stroke patients further confirmed the strength of the method in lesion detection. When compared with the previous version, the current approach showed a higher sensitivity in detecting small lesions and had less false positives around the ventricle and the edge of the brain

The role of corticospinal tract damage in chronic motor recovery and neurorehabilitation: a pilot study

Background. With diffusion-tensor imaging (DTi) it is possible to estimate the structural characteristics of fiber bundles in vivo. This study used DTi to infer damage to the corticospinal tract (CST) and relates this parameter to (a) the level of residual motor ability at least 1 year poststroke and (b) the outcome of intensive motor rehabilitation with constraint-induced movement therapy (CIMT). Objective. To explore the role of CST damage in recovery and CIMT efficacy. Methods. Ten patients with low-functioning hemiparesis were scanned and tested at baseline, before and after CIMT. Lesion overlap with the CST was indexed as reduced anisotropy compared with a CST variability map derived from 26 controls. Residual motor ability was measured through the Wolf Motor Function Test (WMFT) and the Motor Activity Log (MAL) acquired at baseline. CIMT benefit was assessed through the pre—post treatment comparison of WMFT and MAL performance. Results. Lesion overlap with the CST correlated with residual motor ability at baseline, with greater deficits observed in patients with more extended CST damage. Infarct volume showed no systematic association with residual motor ability. CIMT led to significant improvements in motor function but outcome was not associated with the extent of CST damage or infarct volume. Conclusion. The study gives in vivo support for the proposition that structural CST damage, not infarct volume, is a major predictor for residual functional ability in the chronic state. The results provide initial evidence for positive effects of CIMT in patients with varying, including more severe, CST damage.