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Here we report a new polyhydroxylated triterpene, 2β,6β,21α-trihydroxyfriedelan-3-one ( 4 ) isolated from the root and stem bark of Dichapetalum albidum A. Chev (Dichapetalaceae), along with six known triterpenoids ( 1 - 3 , 5 , 6 , 8 ), sitosterol-3β- O -D-glucopyranoside ( 9 ), a dipeptide ( 7 ), and a tyramine derivative of coumaric acid ( 10 ). Friedelan-3-one ( 2 ) showed an antimicrobial activity (IC 50 ) of 11.40 μg/mL against Bacillus cereus , while friedelan-3α-ol ( 1 ) gave an IC 50 of 13.07 μg/mL against Staphylococcus aureus with ampicillin reference standard of 19.52 μg/mL and 0.30 μg/mL respectively. 3β-Acetyl tormentic acid ( 5 ) showed an IC 50 of 12.50 μg/mL against Trypanosoma brucei brucei and sitosterol-3β- O -d-glucopyranoside ( 9 ) showed an IC 50 of 5.06 μg/mL against Leishmania donovani with respective reference standards of IC 50 5.02 μg/mL for suramin and IC 50 0.27 μg/mL for amphotericin B. Molecular docking of the isolated compounds on the enzyme glucose-6-phosphate dehydrogenase (G6PDH) suggested 3β-acetyl tormentic acid ( 5 ) and sitosterol-3β- O -D-glucopyranoside ( 9) as plausible inhibitors of the enzyme in accordance with the experimental biological results observed., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 Published by Elsevier Ltd.)

The function-optimized properties of biominerals arise from the hierarchical organization of primary building blocks. Alteration of properties in response to environmental stresses generally involves time-intensive processes of resorption and reprecipitation of mineral in the underlying organic scaffold. Here, we report that the load-bearing shells of the brachiopod Discinisca tenuis are an exception to this process. These shells can dynamically modulate their mechanical properties in response to a change in environment, switching from hard and stiff when dry to malleable when hydrated within minutes. Using ptychographic X-ray tomography, electron microscopy and spectroscopy, we describe their hierarchical structure and composition as a function of hydration to understand the structural motifs that generate this adaptability. Key is a complementary set of structural modifications, starting with the swelling of an organic matrix on the micron level via nanocrystal reorganization and ending in an intercalation process on the molecular level in response to hydration., (© 2021. The Author(s).)

Collagen fibrils are central to the molecular organization of the extracellular matrix (ECM) and to defining the cellular microenvironment. Glycation of collagen fibrils is known to impact on cell adhesion and migration in the context of cancer and in model studies, glycation of collagen molecules has been shown to affect the binding of other ECM components to collagen. Here we use TEM to show that ribose-5-phosphate (R5P) glycation of collagen fibrils - potentially important in the microenvironment of actively dividing cells, such as cancer cells - disrupts the longitudinal ordering of the molecules in collagen fibrils and, using KFM and FLiM, that R5P-glycated collagen fibrils have a more negative surface charge than unglycated fibrils. Altered molecular arrangement can be expected to impact on the accessibility of cell adhesion sites and altered fibril surface charge on the integrity of the extracellular matrix structure surrounding glycated collagen fibrils. Both effects are highly relevant for cell adhesion and migration within the tumour microenvironment.

Sensitivity enhancement by isotope enrichment and DNP NMR enables detection of minor but biologically relevant species in native intact bone, including nucleic acids, choline from phospholipid headgroups, and histidinyl and hydroxylysyl groups. Labelled matrix from the aggressive osteosarcoma K7M2 cell line confirms the assignments of nucleic acid signals arising from purine, pyrimidine, ribose, and deoxyribose species. Detection of these species is an important and necessary step in elucidating the atomic level structural basis of their functions in intact tissue., Competing Interests: There are no conflicts of interest. Animal procedures were unregulated in terms of the U. K. Animals (Scientific Procedures) Act 1986, but nevertheless were reviewed for compliance with institutional ethical guidelines by the Animal Welfare Ethical Review Board, University of Cambridge., (This journal is © The Royal Society of Chemistry.)

Biomineralization of the extracellular matrix is an essential, regulated process. Inappropriate mineralization of bone and the vasculature has devastating effects on patient health, yet an integrated understanding of the chemical and cell biological processes that lead to mineral nucleation remains elusive. Here, we report that biomineralization of bone and the vasculature is associated with extracellular poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerases in response to oxidative and/or DNA damage. We use ultrastructural methods to show poly(ADP-ribose) can form both calcified spherical particles, reminiscent of those found in vascular calcification, and biomimetically calcified collagen fibrils similar to bone. Importantly, inhibition of poly(ADP-ribose) biosynthesis in vitro and in vivo inhibits biomineralization, suggesting a therapeutic route for the treatment of vascular calcifications. We conclude that poly(ADP-ribose) plays a central chemical role in both pathological and physiological extracellular matrix calcification., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The sparse but functionally essential post-translational collagen modification 5-hydroxylysine can undergo further transformations, including crosslinking, O-glycosylation, and glycation. Dynamic nuclear polarization (DNP) and stable isotope enriched lysine incorporation provide sufficient solid-state NMR sensitivity to identify these adducts directly in skin and vascular smooth muscle cell extracellular matrix (ECM), without extraction procedures, by comparison with chemical shifts of model compounds. Thus, DNP provides access to the elucidation of structural consequences of collagen modifications in intact tissue.

Octacalcium phosphate (OCP; Ca 8 (HPO 4 ) 2 (PO 4 ) 4 . 5H 2 O) is a plausible precursor phase of biological hydroxyapatite, which composites with a number of biologically relevant organic metabolites. Widely used material science physicochemical structure determination techniques successfully characterize the mineral component of these composites but leave details of the structure, and interactions with mineral, of the organic component almost completely obscure. The metabolic linear di-acids succinate (SUC) and adipate (ADI) differentially expand the hydrated (100) layer of OCP. 13 C 13 C correlation (proton driven spin diffusion, PDSD) experiments on OCP composited with (U- 13 C 4 )-SUC, and (U 13 C 6 )-ADI, show that the two di-acids per unit cell adopt non-centrosymmetric but mutually identical structures. 13 C{ 31 P}, rotational echo double resonance (REDOR) shows that one end of each linear di-acid is displaced further from the surface of the apatitic OCP layer relative to the other end. Overall the results indicate two di-acids per unit cell disposed perpendicularly across the OCP hydrated layer with one carboxylate of each di-acid substituting a hydrated surface OCP phosphate group. This study re-affirms the unique advantages of ssNMR in elucidating structural details of organic-inorganic biocomposites, and thereby mechanisms underlying the roles of small metabolites in influencing biomineralization mechanisms and outcomes., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The evolution of assortative mating is a key part of the speciation process. Stronger assortment, or greater divergence in mating traits, between species pairs with overlapping ranges is commonly observed, but possible causes of this pattern of reproductive character displacement are difficult to distinguish. We use a multidisciplinary approach to provide a rare example where it is possible to distinguish among hypotheses concerning the evolution of reproductive character displacement. We build on an earlier comparative analysis that illustrated a strong pattern of greater divergence in penis form between pairs of sister species with overlapping ranges than between allopatric sister-species pairs, in a large clade of marine gastropods (Littorinidae). We investigate both assortative mating and divergence in male genitalia in one of the sister-species pairs, discriminating among three contrasting processes each of which can generate a pattern of reproductive character displacement: reinforcement, reproductive interference and the Templeton effect. We demonstrate reproductive character displacement in assortative mating, but not in genital form between this pair of sister species and use demographic models to distinguish among the different processes. Our results support a model with no gene flow since secondary contact and thus favor reproductive interference as the cause of reproductive character displacement for mate choice, rather than reinforcement. High gene flow within species argues against the Templeton effect. Secondary contact appears to have had little impact on genital divergence.

Fibrillar collagens have mechanical and biological roles, providing tissues with both tensile strength and cell binding sites which allow molecular interactions with cell-surface receptors such as integrins. A key question is: how do collagens allow tissue flexibility whilst maintaining well-defined ligand binding sites? Here we show that proline residues in collagen glycine-proline-hydroxyproline (Gly-Pro-Hyp) triplets provide local conformational flexibility, which in turn confers well-defined, low energy molecular compression-extension and bending, by employing two-dimensional 13 C- 13 C correlation NMR spectroscopy on 13 C-labelled intact ex vivo bone and in vitro osteoblast extracellular matrix. We also find that the positions of Gly-Pro-Hyp triplets are highly conserved between animal species, and are spatially clustered in the currently-accepted model of molecular ordering in collagen type I fibrils. We propose that the Gly-Pro-Hyp triplets in fibrillar collagens provide fibril "expansion joints" to maintain molecular ordering within the fibril, thereby preserving the structural integrity of ligand binding sites.

[This corrects the article DOI: 10.1371/journal.pone.0188955.].

Boosted Regression Trees. Excellent for Data-Poor Spatial Management but Hard to Use: Marine resource managers and scientists often advocate spatial approaches to manage data-poor species. Existing spatial prediction and management techniques are either insufficiently robust, struggle with sparse input data, or make suboptimal use of multiple explanatory variables. Boosted Regression Trees feature excellent performance and are well suited to modelling the distribution of data-limited species, but are extremely complicated and time-consuming to learn and use, hindering access for a wide potential user base and therefore limiting uptake and usage., Brts Automated and Simplified for Accessible General Use With Rich Feature Set: We have built a software suite in R which integrates pre-existing functions with new tailor-made functions to automate the processing and predictive mapping of species abundance data: by automating and greatly simplifying Boosted Regression Tree spatial modelling, the gbm.auto R package suite makes this powerful statistical modelling technique more accessible to potential users in the ecological and modelling communities. The package and its documentation allow the user to generate maps of predicted abundance, visualise the representativeness of those abundance maps and to plot the relative influence of explanatory variables and their relationship to the response variables. Databases of the processed model objects and a report explaining all the steps taken within the model are also generated. The package includes a previously unavailable Decision Support Tool which combines estimated escapement biomass (the percentage of an exploited population which must be retained each year to conserve it) with the predicted abundance maps to generate maps showing the location and size of habitat that should be protected to conserve the target stocks (candidate MPAs), based on stakeholder priorities, such as the minimisation of fishing effort displacement., Gbm.auto for Management in Various Settings: By bridging the gap between advanced statistical methods for species distribution modelling and conservation science, management and policy, these tools can allow improved spatial abundance predictions, and therefore better management, decision-making, and conservation. Although this package was built to support spatial management of a data-limited marine elasmobranch fishery, it should be equally applicable to spatial abundance modelling, area protection, and stakeholder engagement in various scenarios.

We have prepared mouse fur extensively 13 C, 15 N-labelled in all amino acid types enabling application of 2D solid state NMR techniques which establish covalent and spatial proximities within, and in favorable cases between, residues. 13 C double quantum-single quantum correlation and proton driven spin diffusion techniques are particularly useful for resolving certain amino acid types. Unlike 1D experiments on isotopically normal material, the 2D methods allow the chemical shifts of entire spin systems of numerous residue types to be determined, particularly those with one or more distinctively shifted atoms such as Gly, Ser, Thr, Tyr, Phe, Val, Leu, Ile and Pro. Also the partial resolution of the amide signals into two signal envelopes comprising of α-helical, and β-sheet/random coil components, enables resolution of otherwise overlapped α-carbon signals into two distinct cross peak families corresponding to these respective secondary structural regions. The increase in resolution conferred by extensive labelling offers new opportunities to study the chemical fate and structural environments of specific atom and amino acid types under the influence of commercial processes, and therapeutic or cosmetic treatments., Competing Interests: Compliance with ethical standards Conflicts of interest None of the authors have any conflicts of interest in regard to this work. Human and animal rights No human subjects or tissues were used. All animal procedures complied with institutional ethical guidelines, and the U. K. Animals (Scientific Procedures) Act 1986.

Structural biominerals are inorganic/organic composites that exhibit remarkable mechanical properties. However, the structure-property relationships of even the simplest building unit-mineral single crystals containing embedded macromolecules-remain poorly understood. Here, by means of a model biomineral made from calcite single crystals containing glycine (0-7 mol%) or aspartic acid (0-4 mol%), we elucidate the origin of the superior hardness of biogenic calcite. We analysed lattice distortions in these model crystals by using X-ray diffraction and molecular dynamics simulations, and by means of solid-state nuclear magnetic resonance show that the amino acids are incorporated as individual molecules. We also demonstrate that nanoindentation hardness increased with amino acid content, reaching values equivalent to their biogenic counterparts. A dislocation pinning model reveals that the enhanced hardness is determined by the force required to cut covalent bonds in the molecules.

Fluorescein is known to exist in three tautomeric forms defined as quinoid, zwitterionic, and lactoid. In the solid state, the quinoid and zwitterionic forms give rise to red and yellow materials, respectively. The lactoid form has not been crystallized pure, although its cocrystal and solvate forms exhibit colors ranging from yellow to green. An explanation for the observed colors of the crystals is found using a combination of UV/Vis spectroscopy and plane-wave DFT calculations. The role of cocrystal coformers in modifying crystal color is also established. Several new crystal structures are determined using a combination of X-ray and electron diffraction, solid-state NMR spectroscopy, and crystal structure prediction (CSP). The protocol presented herein may be used to predict color properties of materials prior to their synthesis., (© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Members of the genus Pirenella are abundant inhabitants of intertidal sedimentary shores, often found in association with mangroves, on the continental margins of the western Pacific and Indian Oceans, and eastern Mediterranean Sea. Until recently, four morphological species were recognised in the tropical Indo-West Pacific region and classified in the genus Cerithideopsilla, while another species occupying the Mediterranean and Indian Ocean was classified as Pirenella conica. Molecular phylogenetic analysis has demonstrated that all these species are congeneric and here it is shown that the valid name for the genus is Pirenella. A recently published molecular study recognised a total of 16 species and the present work is a systematic account of these species. Of the 16, nine are described as new. Other significant nomenclatural acts are: fixation of type species of Pirenella as Pirenella mammillata J.E. Gray, 1847; designation of neotypes for Cerithium alatum Philippi, 1849, Cerithium microptera Kiener, 1841, Cerithium conicum Blainville, 1829, Pirenella mammillata J.E. Gray, 1847 and Murex cingulatus Gmelin, 1791; designation of lectotype for Cerithium retiferum G.B. Sowerby II, 1855. The species accounts include full synonymies, detailed descriptions of shells (based on 831 museum samples), distribution records and maps, reviews of life history, of habitat and of ecology, and some images of radulae. Details of shell sculpture are adequate for the diagnosis of most species. Distorted shells are common in some populations and are suggested to represent parasitised individuals. Some species are pests of fishponds in Southeast Asia and P. conica is the intermediate host of a trematode responsible for the human disease heterophyiasis, while others are threatened by habitat destruction.

The mechanism and products of the structural collapse of the metal–organic frameworks (MOFs) UiO-66, MIL-140B and MIL-140C upon ball-milling are investigated through solid state 13C NMR and pair distribution function (PDF) studies, finding amorphization to proceed by the breaking of a fraction of metal–ligand bonding in each case. The amorphous products contain inorganic–organic bonding motifs reminiscent of the crystalline phases. Whilst the inorganic Zr6O4(OH)4 clusters of UiO-66 remain intact upon structural collapse, the ZrO backbone of the MIL-140 frameworks undergoes substantial distortion. Density functional theory calculations have been performed to investigate defective models of MIL-140B and show, through comparison of calculated and experimental 13C NMR spectra, that amorphization and defects in the materials are linked.

An appreciable level of isotope labelling is essential for future NMR structure elucidation of mammalian biomaterials, which are either poorly expressed, or unexpressable, using micro-organisms. We present a detailed protocol for high level (13)C enrichment even in slow turnover murine biomaterials (fur keratin), using a customized diet supplemented with commercial labelled algal hydrolysate and formulated as a gel to minimize wastage, which female mice consumed during pregnancy and lactation. This procedure produced approximately eightfold higher fur keratin labelling in pups, exposed in utero and throughout life to label, than in adults exposed for the same period, showing both the effectiveness, and necessity, of this approach.

Collagens, the most abundant proteins in mammals, are defined by their triple-helical structures and distinctive Gly-Xaa-Yaa repeating sequence, where Xaa is often proline and Yaa, hydroxyproline (Hyp/O). It is known that hydroxyproline in the Yaa position stabilises the triple helix, and that lack of proline hydroxylation in vivo leads to dysfunctional collagen extracellular matrix assembly, due to a range of factors such as a change in hydration properties. In addition, we note that in model peptides, when Yaa is unmodified proline, the Xaa proline has a strong propensity to adopt an endo ring conformation, whilst when Yaa is hydroxyproline, the Xaa proline adopts a range of endo and exo conformations. Here we use a combination of solid-state NMR spectroscopy and potential energy landscape modelling of synthetic triple-helical collagen peptides to understand this effect. We show that hydroxylation of the Yaa proline causes the Xaa proline ring conformation to become metastable, which in turn confers flexibility on the triple helix.

We have developed a strategy for selective imaging of collagen in live foetal ovine osteoblasts. Our approach involves the incorporation of an azide-tagged proline in the biosynthesis of collagen followed by labelling using a strain-promoted [3+2] azide-alkyne cycloaddition reaction.

Public concern over biodiversity loss is often rationalized as a threat to ecosystem functioning, but biodiversity-ecosystem functioning (BEF) relations are hard to empirically quantify at large scales. We use a realistic marine food-web model, resolving species over five trophic levels, to study how total fish production changes with species richness. This complex model predicts that BEF relations, on average, follow simple Michaelis-Menten curves when species are randomly deleted. These are shaped mainly by release of fish from predation, rather than the release from competition expected from simpler communities. Ordering species deletions by decreasing body mass or trophic level, representing 'fishing down the food web', accentuates prey-release effects and results in unimodal relationships. In contrast, simultaneous unselective harvesting diminishes these effects and produces an almost linear BEF relation, with maximum multispecies fisheries yield at ≈40% of initial species richness. These findings have important implications for the valuation of marine biodiversity.

Nuclear magnetic resonance (NMR) spectroscopy is useful to determine molecular structure in tissues grown in vitro only if their fidelity, relative to native tissue, can be established. Here, we use multidimensional NMR spectra of animal and in vitro model tissues as fingerprints of their respective molecular structures, allowing us to compare the intact tissues at atomic length scales. To obtain spectra from animal tissues, we developed a heavy mouse enriched by about 20% in the NMR-active isotopes carbon-13 and nitrogen-15. The resulting spectra allowed us to refine an in vitro model of developing bone and to probe its detailed structure. The identification of an unexpected molecule, poly(adenosine diphosphate ribose), that may be implicated in calcification of the bone matrix, illustrates the analytical power of this approach., (Copyright © 2014, American Association for the Advancement of Science.)

We provide evidence that citrate anions bridge between mineral platelets in bone and hypothesize that their presence acts to maintain separate platelets with disordered regions between them rather than gradual transformations into larger, more ordered blocks of mineral. To assess this hypothesis, we take as a model for a citrate bridging between layers of calcium phosphate mineral a double salt octacalcium phosphate citrate (OCP-citrate). We use a combination of multinuclear solid-state NMR spectroscopy, powder X-ray diffraction, and first principles electronic structure calculations to propose a quantitative structure for this material, in which citrate anions reside in a hydrated layer, bridging between apatitic layers. To assess the relevance of such a structure in native bone mineral, we present for the first time, to our knowledge, (17)O NMR data on bone and compare them with (17)O NMR data for OCP-citrate and other calcium phosphate minerals relevant to bone. The proposed structural model that we deduce from this work for bone mineral is a layered structure with thin apatitic platelets sandwiched between OCP-citrate-like hydrated layers. Such a structure can explain a number of known structural features of bone mineral: the thin, plate-like morphology of mature bone mineral crystals, the presence of significant quantities of strongly bound water molecules, and the relatively high concentration of hydrogen phosphate as well as the maintenance of a disordered region between mineral platelets.

NMR is ideal for characterizing non-enzymatic protein glycation, including AGEs (advanced glycation endproducts) underlying tissue pathologies in diabetes and ageing. Ribose, R5P (ribose-5-phosphate) and ADPR (ADP-ribose), could be significant and underinvestigated biological glycating agents especially in chronic inflammation. Using [U- 13 C]ribose we have identified a novel glycoxidation adduct, 5-deoxy-5-desmethylpronyl-lysine, 'norpronyl-lysine', as well as numerous free ketones, acids and amino group reaction products. Glycation by R5P and ADPR proceeds rapidly with R5P generating a brown precipitate with PLL (poly-L-lysine) within hours. ssNMR (solid-state NMR) 13 C- 13 C COSY identifies several crosslinking adducts such as the newly identified norpronyl-lysine, in situ, from the glycating reaction of 13 C 5 -ribose with collagen. The same adducts are also identifiable after reaction of collagen with R5P. We also demonstrate for the first time bio-amine (spermidine, N-acetyl lysine, PLL) catalysed ribose 2-epimerization to arabinose at physiological pH. This work raises the prospect of advancing understanding of the mechanisms and consequences of glycation in actual tissues, in vitro or even ex vivo, using NMR isotope-labelled glycating agents, without analyses requiring chemical or enzymatic degradations, or prior assumptions about glycation products., (© 2014 The author(s).)

The genus Cerithideopsis is most common in mangrove and salt marsh habitats of the New World tropics, but there is also a small radiation in the Indo-West Pacific region. Previously, these Indo-Pacific snails have generally been classified as Cerithidea largillierti (Philippi, 1848). Molecular phylogenetic analysis (partial sequences of mitochondrial COI and 16S rRNA, and nuclear 28S rRNA) of 15 specimens from 8 localities between Japan and Australia reveal three clades, among which there are small morphological differences and which show allopatric distributions. Cerithideopsis largillierti sensu stricto is restricted to Japan and China, while the two other species are described as new: C. australiensis occurs in tropical Australasia and C. malayensis is found from Malaysia to Java and the Philippines. All occur on mud and in pools with leaf litter, in the shaded landward and middle zones of mangrove forests, and do not climb the trees. The species accounts include full synonymies, detailed descriptions of shells based on 82 museum samples, descriptions of living animals, distribution records and maps, and notes on habitat and conservation status.

Members of the genus Cerithidea are common components of the fauna of mangrove forests, tidal swamps and salt marshes in the Indo-West Pacific province, in marine and brackish conditions. The snails typically rest on the trunks of trees and other vegetation and migrate to the substrate to feed at low tide. In many areas mangrove habitats are under threat and some Cerithidea species are therefore considered to be endangered. In current taxonomic literature, ten species are recognized on the basis of shell morphology. A recently published molecular phylogenetic analysis has increased this to fifteen. The present study is a systematic account of these fifteen recognized species. Of these, two are newly described: C. houbricki and C. andamanensis. The species accounts include full synonymies, detailed descriptions of shells based on 621 museum samples, descriptions of living animals, distribution records and maps, reviews of habitat and ecology, and notes on conservation status.

Understanding the pressures of fisheries on the ecosystem is crucial for effective management. Fishery removals, or catch, are composed of both landings and discards. However, the use of discards data in studies investigating the effect of the fishing pressures is sparse. Here, we explore the individual contribution of both these catch components to the overall pressure of fisheries on the ecosystem metrics. Using Irish observer data, we compare the linear relationship between several ecological metrics calculated for landings and discards with those of catch. Our results show that in fisheries with high discarding rates, discards can drive the fisheries' ecological fingerprint and highlight the need to rectify landings-based estimates to make them representative of those of catch in order to gain a robust picture of the impact of fisheries.

(13)C NMR is an effective method of characterizing proanthocyanidin (PAC) tannins in quebracho (Schinopsis lorentzii) heartwood and black wattle (Acacia mearnsii) bark, before and after commercial extraction. The B-rings of the constituent flavan-3-ols, catechols (quebracho) or pyrogallols (wattle), are recognized in unprocessed source materials by "marker" signals at ca. 118 or 105ppm, respectively. NMR allows the minimum extraction efficiency to be calculated; ca. 30%, and ca. 80%, for quebracho heartwood and black wattle bark, respectively. NMR can also identify PAC tannin (predominantly robinetinidin), and compare tannin content, in bark from other acacia species; tannin content decreases in the order A. mearnsii, Acacia pycnantha (87% of A. mearnsii), Acacia dealbata and Acacia decurrens (each 74%) and Acacia karroo (30%). Heartwood from an underexploited PAC tannin source, Searsia lancea, taxonomically close to quebracho, shows abundant profisetinidin and catechin PACs. NMR offers the advantage of being applicable to source materials in their native state, and has potential applications in optimizing extraction processes, identification of tannin sources, and characterization of tannin content in cultivar yield improvement programmes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

There is continuing debate about whether abundant citrate plays an active role in biomineralization of bone. Using solid state NMR dipolar dephasing, we examined another normally mineralized hard tissue, mineralized articular cartilage, as well as biocalcifications arising in pathological conditions, mineralized intimal atherosclerotic vascular plaque, and apatitic uroliths (urinary stones). Residual nondephasing ¹³C NMR signal at 76 ppm in the spectra of mineralized cartilage and vascular plaque indicates that a quaternary carbon atom resonates at this frequency, consistent with the presence of citrate. The presence, and as yet unproven possible mechanistic involvement, of citrate in tissue mineralization extends the compositional, structural, biogenetic, and cytological similarities between these tissues and bone itself. Out of 10 apatitic kidney stones, five contained NMR-detectable citrate. Finding citrate in a high proportion of uroliths may be significant in view of the use of citrate in urolithiasis therapy and prophylaxis. Citrate may be essential for normal biomineralization (e.g., of cartilage), play a modulatory role in vascular calcification which could be a target for therapeutic intervention, and drive the formation of apatitic rather than other calcific uroliths, including more therapeutically intractable forms of calcium phosphate.

The monophyly of the muricid subfamily Rapaninae has recently been confirmed with molecular techniques, but its composition and the relationships among its constituent genera remain unclear. We use four genes (28S rRNA, 12S rRNA, 16S rRNA and cytochrome c oxidase subunit I, COI) to construct a Bayesian phylogeny of 80 rapanine species (73% of the approximately 109 currently accepted), representing 27 of the 31 nominal genera. This is the most complete phylogeny of this taxonomically confusing subfamily yet produced. We propose a revised phylogenetic classification of the Rapaninae, assigning the recognized species to 28 genera. Most of the morphologically-defined rapanine genera are considered valid, including Purpura, Drupa, Thais and Nassa, but many of them are here restricted or redefined so that they are monophyletic. In particular the familiar genus Thais is narrowly restricted to a single species. Many groups previously accepted as subgenera, including Mancinella, Vasula, Thalessa and Thaisella, are here accorded full generic rank. We describe one new genus, Indothais. While we do not formally alter species-level taxonomy, we show molecular evidence for two cryptic species and several instances of probable species synonymy. We estimate the age of diversification of the Rapaninae as Late Cretaceous (75.9 Ma) and of many of its genera as Miocene., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Pathomechanisms underlying vascular calcification biogenesis are still incompletely understood. Biomineral from human atherosclerotic intimal plaques; human, equine, and bovine medial vascular calcifications; and human and equine bone was released from collagenous organic matrix by sodium hydroxide/sodium hypochlorite digestion. Solid-state (13)C NMR of intimal plaque mineral shows signals from cholesterol/cholesteryl esters and fatty acids. In contrast, in mineral from pure medial calcifications and bone mineral, fatty acid signals predominate. Refluxing (chloroform/methanol) intimal plaque calcifications removes the cholesterylic but not the fatty acyl signals. The lipid composition of this refluxed mineral now closely resembles that of the medial and bone mineral, which is unchanged by reflux. Thus, intimal and medial vascular calcifications and bone mineral have in common a pool of occluded mineral-entrained fatty acyl-rich lipids. This population of fatty acid may contain methyl-branched fatty acids, possibly representing lipoprotein particle remnants. Cell signaling and mechanistic parallels between physiological (orthotopic) and pathological (ectopic) calcification are also reflected thus in the NMR spectroscopic fingerprints of mineral-associated and mineral-entrained lipids. Additionally the atherosclerotic plaque mineral alone shows a significant independent pool of cholesterylic lipids. Colocalization of mineral and lipid may be coincidental, but it could also reflect an essential mechanistic component of biomineralization.

Ibliform barnacles are among the few invertebrate animals harnessing calcium phosphate to construct hard tissue. The (31)P solid-state NMR (SSNMR) signal from the shell plates of Ibla cumingi (Iblidae) is broader than that of bone, and shifted by ca 1 ppm to low frequency. (1)H-(31)P heteronuclear correlation (HETCOR) experiments show a continuum of different phosphorus/phosphate atomic environments, close to hydrogen populations with resonance frequencies between ca 10 and 20 ppm. Associated (1)H and (31)P chemical shifts argue the coexistence of weakly (high (31)P frequency, low (1)H frequency) to more strongly (lower (31)P frequency, higher (1)H frequency) hydrogen-bonded hydrogen phosphate-like molecular/ionic species. There is no resolved signal from discrete OH(-) ions. (13)C SSNMR shows chitin, protein and other organic biomolecules but, unlike bone, there are no significant atomic scale organic matrix-mineral contacts. The poorly ordered hydrogen phosphate-like iblid mineral is strikingly different, structurally and compositionally, from both vertebrate bone mineral and the more crystalline fluoroapatite of the linguliform brachiopods. It probably represents a previously poorly characterized calcium phosphate biomineral, the evolution of which may have reflected either the chemical conditions of ancestral seas or the mechanical advantages of phosphatic biomineralization over a calcium carbonate equivalent.

Snails in the closely related trochid genera Phorcus Risso, 1826 and Osilinus Philippi, 1847 are ecologically important algal grazers in the intertidal zone of the northeastern Atlantic Ocean and Mediterranean Sea. Here we present the first complete molecular phylogeny for these genera, based on the nuclear 28S rRNA gene and the mitochondrial 16S rRNA and COI genes, and show that the current classification is erroneous. We recognize nine species in a single genus, Phorcus: estimated by BEAST analysis, this arose 30 (± 10) Ma; it consists of two subgenera, Phorcus and Osilinus, which we estimate diverged 14 (± 4.5) Ma. Osilinus kotschyi, from the Arabian and Red Seas, is not closely related and is tentatively referred to Priotrochus Fischer, 1879. Our phylogeny allows us to address biogeographical questions concerning the origins of the Mediterranean and Macaronesian species of this group. The former appear to have evolved from Atlantic ancestors that invaded the Mediterranean on several occasions after the Zanclean Flood, which ended the Messinian Salinity Crisis 5.3 Ma; whereas the latter arose from several colonizations of mainland Atlantic ancestors within the last 3 (± 1.5) Ma., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The life history characteristics of some elasmobranchs make them particularly vulnerable to fishing mortality; about a third of all species are listed by the IUCN as Threatened or Near Threatened. Marine Protected Areas (MPAs) have been suggested as a tool for conservation of elasmobranchs, but they are likely to be effective only if such populations respond to fishing impacts at spatial-scales corresponding to MPA size. Using the example of the Celtic Sea, we modelled elasmobranch biomass (kg h(-1)) in fisheries-independent survey hauls as a function of environmental variables and 'local' (within 20 km radius) fishing effort (h y(-1)) recorded from Vessel Monitoring Systems data. Model selection using AIC suggested strongest support for linear mixed effects models in which the variables (i) fishing effort, (ii) geographic location and (iii) demersal fish assemblage had approximately equal importance in explaining elasmobranch biomass. In the eastern Celtic Sea, sampling sites that occurred in the lowest 10% of the observed fishing effort range recorded 10 species of elasmobranch including the critically endangered Dipturus spp. The most intensely fished 10% of sites had only three elasmobranch species, with two IUCN listed as Least Concern. Our results suggest that stable spatial heterogeneity in fishing effort creates de facto refugia for elasmobranchs in the Celtic Sea. However, changes in the present fisheries management regime could impair the refuge effect by changing fisher's behaviour and displacing effort into these areas.

Unlabelled: In pilot studies of the usefulness of solid state nuclear magnetic resonance spectroscopy in characterizing chemical and molecular structural effects of alkaptonuria on connective tissue, we have obtained (13) C spectra from articular cartilage from an AKU patient. An apparently normal anatomical location yielded a cross polarization magic angle spinning spectrum resembling literature spectra and dominated by collagen and glycosaminoglycan signals. All spectral linewidths from strongly pigmented ochronotic cartilage however were considerably increased relative to the control indicating a marked increase in collagen molecular disorder. This disordering of cartilage structural protein parallels, at the atomic level, the disordering revealed at higher length scales by microscopy. We also demonstrate that the abnormal spectra from ochronotic cartilage fit with the abnormality in the structure of collagen fibres at the ultrastructural level, whereby large ochronotic deposits appear to alter the structure of the collagen fibre by invasion and cross linking., Summary: Increased signal linewidths in solid state NMR spectra of ochronotic articular cartilage from an AKU patient relative to linewidths in normal, control, cartilage reveals a marked decrease in collagen molecular order in the diseased tissue. This atomic level disordering parallels higher length scale disorder revealed by microscopic techniques.

Abalones ( Haliotis species) are a popular delicacy and commonly preserved in dried form either whole or in slices or small pieces for consumption in Asian countries. Driven by the huge profit from trading abalones, dishonest traders may substitute other molluscan species for processed abalone, of which the morphological characteristics are frequently lost in the processed form. For protection of consumer rights and law enforcement against fraud, there is a need for an effective methodology to differentiate between fake and genuine abalone. This paper describes a method (validated according to the international forensic guidelines provided by SWGDAM) for the identification of fake abalone species using forensically informative nucleotide sequence (FINS) analysis. A study of the local market revealed that many claimed "abalone slice" samples on sale are not genuine. The fake abalone samples were found to be either volutids of the genus Cymbium (93%) or the muricid Concholepas concholepas (7%). This is the first report of Cymbium species being used for the preparation and sale as "abalone" in dried sliced form in Hong Kong.

Unusually for invertebrates, linguliform brachiopods employ calcium phosphate mineral in hard tissue formation, in common with the evolutionarily distant vertebrates. Using solid-state nuclear magnetic resonance spectroscopy (SSNMR) and X-ray powder diffraction, we compare the organic constitution, crystallinity and organic matrix-mineral interface of phosphatic brachiopod shells with those of vertebrate bone. In particular, the organic-mineral interfaces crucial for the stability and properties of biomineral were probed with SSNMR rotational echo double resonance (REDOR). Lingula anatina and Discinisca tenuis shell materials yield strikingly dissimilar SSNMR spectra, arguing for quite different organic constitutions. However, their fluoroapatite-like mineral is highly crystalline, unlike the poorly ordered hydroxyapatite of bone. Neither shell material shows (13)C{(31)P} REDOR effects, excluding strong physico-chemical interactions between mineral and organic matrix, unlike bone in which glycosaminoglycans and proteins are composited with mineral at sub-nanometre length scales. Differences between organic matrix of shell material from L. anatina and D. tenuis, and bone reflect evolutionary pressures from contrasting habitats and structural purposes. The absence of organic-mineral intermolecular associations in brachiopod shell argues that biomineralization follows different mechanistic pathways to bone; their details hold clues to the molecular structural evolution of phosphatic biominerals, and may provide insights into novel composite design.

Purpose: We characterized the biomacromolecular composition of phosphatic urinary stones using solid state nuclear magnetic resonance spectroscopy. We identified possible parallels between the nature of the organic matrix-mineral interface in stones and that in other mineralized tissue using nuclear magnetic resonance spectroscopy rotational echo double resonance., Materials and Methods: We analyzed 28 phosphatic (apatite and mixed apatite-struvite) surgically removed stones by nuclear magnetic resonance spectroscopy using (31)P, (13)C and a 9.4 Tesla magnetic field. Ten samples had sufficient signal from biomacromolecular organic material to characterize the mineral/organic interface by (13)C{(31)P} rotational echo double resonance., Results: Biomacromolecular organic material was most abundant in phosphatic stones in which apatite predominated. Nuclear magnetic resonance spectroscopy detected variable proportions of protein, glycosaminoglycan, lipid and carbonate. Rotational echo double resonance revealed strong interaction between mineral and glycosaminoglycan molecules, and to a lesser extent protein molecules, on the sub-nm length scale, implying that glycosaminoglycan and protein are composited into or onto the mineral lattice by strong physicochemical interactions. Carbonate ions substituted into apatite crystal lattices also showed the expected strong (13)C{(31)P} rotational echo double resonance effects. Conversely when present, lipid, calcium oxalate hydrates and uric acid showed no rotational echo double resonance effects, proving that they exist as deposits or crystals distinct from phosphatic mineral/biomacromolecular composites., Conclusions: The intimate coexistence of biomacromolecules, especially glycosaminoglycan, with apatite in phosphatic stones supports the notion that they may have a key role in stone pathogenesis. The underlying intermolecular relationships may reflect those governing the formation of Randall's plaque in nascent stones., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Solid state ¹³C-NMR spectra of pure tannin powders from four different sources--mimosa, quebracho, chestnut and tara--are readily distinguishable from each other, both in pure commercial powder form, and in leather which they have been used to tan. Groups of signals indicative of the source, and type (condensed vs. hydrolyzable) of tannin used in the manufacture are well resolved in the spectra of the finished leathers. These fingerprints are compared with those arising from leathers tanned with other common tanning agents. Paramagnetic chromium (III) tanning causes widespread but selective disappearance of signals from the spectrum of leather collagen, including resonances from acidic aspartyl and glutamyl residues, likely bound to Cr (III) structures. Aluminium (III) and glutaraldehyde tanning both cause considerable leather collagen signal sharpening suggesting some increase in molecular structural ordering. The ²⁷Al-NMR signal from the former material is consistent with an octahedral coordination by oxygen ligands. Solid state NMR thus provides easily recognisable reagent specific spectral fingerprints of the products of vegetable and some other common tanning processes. Because spectra are related to molecular properties, NMR is potentially a powerful tool in leather process enhancement and quality or provenance assurance.

We present a proof-of-principle study of an environmentally-friendly approach to laboratory research, in which the synthesis and structural characterisation of metal-organic complexes and frameworks are achieved without using bulk solvents; our study addresses the use of heteroditopic ligands for manipulating the dimensionality of metal-organic materials and describes how kinetic obstacles in such mechanosynthesis can be overcome.

Recognition of bone mineral by bisphosphonates is crucial to their targeting, efficacy, therapeutic and diagnostic applications, and pharmacokinetics. In a search for rapid and simple NMR approaches to assessing the bone recognition characteristics of bisphosphonates, we have studied alendronate, pamidronate, neridronate, zoledronate and tiludronate, in crystalline form and bound to the surface of pure bone mineral stripped of its organic matrix by a simple chemical process. (31)P NMR chemical shift anisotropies and asymmetries in the crystalline compounds cluster strongly into groupings corresponding to fully protonated, monoprotonated, and deprotonated phosphonate states. All the mineral-bound bisphosphonates cluster in the same anisotropy-asymmetry space as the deprotonated phosphonates. In (13)C{(31)P} rotational echo double resonance (REDOR) experiments, which are sensitive to carbon-phosphorus interatomic distances, the strongly mineral-bound alendronate displays very similar conformational and side chain dynamics to its crystalline state. Pamidronate and neridronate, with shorter and longer sidechains, respectively, and generally weaker mineral binding, display more dynamical sidechains in the mineral-bound state. The REDOR experiment provides a simple rationalization of bisphosphonate-mineral affinity in terms of molecular structure and dynamics, consistent with findings from much more labour- and time-intensive isotope labelling approaches., (2010 Elsevier B.V. All rights reserved.)

Marine protected areas (MPAs) are attaining increasing importance in the management of marine ecosystems. They are effective for conservation in tropical and subtropical areas (mainly coral and rocky reefs), but it is debated whether they are useful in the management of migratory fish stocks in open temperate regions. World War II created a large marine area within which commercial fishing was prevented for 6 years. Here we analyse scientific trawl data for three important North Sea gadoids, collected between 1928 and 1958. Using statistical models to summarise the data, we demonstrate the potential of MPAs for expediting the recovery of over-exploited fisheries in open temperate regions. Our age-structured data and population models suggest that wild fish stocks will respond rapidly and positively to reductions in harvesting rates and that the numbers of older fish in a population will react before, and in much greater proportion, than their younger counterparts in a kind of Mexican wave. Our analyses demonstrate both the overall increase in survival due to the lack of harvesting in the War and the form of the age-dependent wave in numbers. We conclude that large closed areas can be very useful in the conservation of migratory species from temperate areas and that older fish benefit fastest and in greater proportion. Importantly, any rise in spawning stock biomass may also not immediately result in better recruitment, which can respond more slowly and hence take longer to contribute to higher future harvestable biomass levels.

Failure to express or expression of dysfunctional low-density lipoprotein receptors (LDLR) causes familial hypercholesterolemia in humans, a disease characterized by elevated blood cholesterol concentrations, xanthomas, and coronary heart disease, providing compelling evidence that high blood cholesterol concentrations cause atherosclerosis. In this study, we used (1)H nuclear magnetic resonance spectroscopy to examine the metabolic profiles of plasma and urine from the LDLR knockout mice. Consistent with previous studies, these mice developed hypercholesterolemia and atherosclerosis when fed a high-fat/cholesterol/cholate-containing diet. In addition, multivariate statistical analysis of the metabolomic data highlighted significant differences in tricarboxylic acid cycle and fatty acid metabolism, as a result of high-fat/cholesterol diet feeding. Our metabolomic study also demonstrates that the effect of high-fat/cholesterol/cholate diet, LDLR gene deficiency, and the diet-genotype interaction caused a significant perturbation in choline metabolism, notably the choline oxidation pathway. Specifically, the loss in the LDLR caused a marked reduction in the urinary excretion of betaine and dimethylglycine, especially when the mice are fed a high-fat/cholesterol/cholate diet. Furthermore, as we demonstrate that these metabolic changes are comparable with those detected in ApoE knockout mice fed the same high-fat/cholesterol/cholate diet they may be useful for monitoring the onset of atherosclerosis across animal models.

We have studied the atomic level structure of mineralized articular cartilage with heteronuclear solid-state NMR, our aims being to identify the inorganic species present at the surfaces of the mineral crystals which may interact with the surrounding organic matrix and to determine which components of the organic matrix are most closely involved with the mineral crystals. One-dimensional (1)H and (31)P and two-dimensional (1)H-(31)P heteronuclear correlation NMR experiments show that the mineral component is very similar to that in bone with regard to its surface structure. (13)C{(31)P} rotational echo double resonance experiments identify the organic molecules at the mineral surface as glycosaminoglycans, which concurs with our recent finding in bone. There is also evidence of gamma-carboxyglutamic acid residues interacting with the mineral. However, other matrix components appear more distant from the mineral compared with bone. This may be due to a larger hydration layer on the mineral crystal surfaces in calcified cartilage.

Objective: Cell biological studies demonstrate remarkable similarities between mineralization processes in bone and vasculature, but knowledge of the components acting to initiate mineralization in atherosclerosis is limited. The molecular level microenvironment at the organic-inorganic interface holds a record of the mechanisms controlling mineral nucleation. This study was undertaken to compare the poorly understood interface in mineralized plaque with that of bone, which is considerably better characterized., Methods and Results: Solid state nuclear magnetic resonance (SSNMR) spectroscopy provides powerful tools for studying the organic-inorganic interface in calcium phosphate biominerals. The rotational echo double resonance (REDOR) technique, applied to calcified human plaque, shows that this interface predominantly comprises sugars, most likely glycosaminoglycans (GAGs). In this respect, and in the pattern of secondary effects seen to protein (mainly collagen), calcified plaque strongly resembles bone., Conclusions: The similarity between biomineral formed under highly controlled (bone) and pathological (plaque) conditions suggests that the control mechanisms are more similar than previously thought, and may be adaptive. It is strong further evidence for regulation of plaque mineralization by osteo/chondrocytic vascular smooth muscle cells.

Geographically separate biotas often show differences in species composition, diversity, and adaptations. Such differences, which often reflect historical differences in regimes of productivity and selection, have received little attention from ecologists. Here we concentrate on diversity and maximum body size in 18 guilds of shallow-water marine shell-bearing mollusks and barnacles from the European and North American sides of the temperate North Atlantic. These guilds represent suspension-feeders (epifaunal, shallow infaunal, and deep infaunal), chemosymbiotic bivalves, predators, and grazers. Geographic patterns among Recent guilds were compared to those during Pliocene and early Miocene time in order to determine how diversity and maximum size changed over the last 17 million years in the face of high levels of extinction and species invasion. Recent European guilds are generally more diverse than their American counterparts, a finding consistent with previous biota-wide analyses. Diversity within Pliocene guilds was often higher on the American side, but this stems in part from the large subtropical component in preserved temperate Pliocene guilds in Virginia but not in Europe. The largest species in hard-bottom guilds in Europe reach greater sizes than those in comparable American guilds, but for sand-bottom guilds, American species almost always attain greater sizes than their European counterparts. These size differences have changed little since early Miocene time despite high levels of Pliocene and Pleistocene extinction in invasion, particularly on the American side. Large-bodied species are overrepresented among both extinct and invading lineages, meaning that lineage replacement has been the rule for the largest species in guilds on both sides of the Atlantic. Together with previous studies, these results may imply that sand-bottom environments in North America have been more productive and have experienced more far-reaching escalation between shell-bearing species and their predators than their European counterparts since early Miocene time. The Pliocene to Recent record of hard-bottom guilds implies that productivity and/or escalation in these systems may have been higher on the European side than in eastern North America. Shallow-water guilds on opposite sides of the Atlantic have retained differences despite great upheavals caused by extinctions and invasions during the last 3 million years.

Magnetic resonance (MR) provides an attractive non-invasive way of studying drug distribution in vivo. Widespread occurrence of fluorine in medicinal compounds, and its favourable MR properties, make it an effective probe for drug absorption, distribution, metabolism and excretion (ADME) studies. We discuss practicalities of detection and localization, and when (19)F MR would add value in a clinical trial, exemplified by deployments in oncology and psychiatry, where it is a practical way of demonstrating chronic brain accumulation directly. Limitations are emphasized to minimize failure risk, for example, inadequate sensitivity relative to tissue drug concentrations. The review anticipates increasing clinical (19)F MR as high field human scanners become widespread, and requirements to demonstrate mechanisms underlying clinical effects become more pressing.

Comparison of structure and hydration stability of pairs of chiral and racemic binary cocrystals indicates that the racemic solid is more stable than the chiral one; we illustrate that this difference might arise from intermolecular (crystal packing) factors in one case, while intramolecular (molecular conformation) factors are more significant in the other.