Seaweed fertilisation impacts the chemical and isotopic composition of barley : Implications for analyses of archaeological skeletal remains

This research was partially funded by the European Social Fund and Scottish Funding Council as part of Developing Scotland's Workforce in the Scotland 2014–2020 European Structural and Investment Fund Programme. The contribution of staff from the University of the Highlands and Islands' Agronomy Institute and the James Hutton Institute to the field trial was supported by Rural and Environment Science and Analytical Services (RESAS) funding from the Scottish Government. GPS geolocation was performed by archaeologists of the Orkney Research Centre for Archaeology (ORCA). Stable isotope ratio measurements were performed at the Scottish Universities Environmental Research Centre (SUERC), East Kilbride, and elemental composition analysis was performed at the Trace Element Speciation Laboratory, Aberdeen (TESLA). MB would like to thank IM's family for their help collecting and storing the decomposing seaweed.Peer reviewedPostprintPostprin

Trace element ratios in tooth enamel as palaeodietary indicators of seaweed consumption and coastal grazing, and their broader applicability

Ratios of barium and strontium concentrations in skeletal samples (e.g. in the logarithmic form lg(Ba/Sr)), are a possible alternative or supplementary marker to stable carbon isotope ratios (δ13C) for identification of marine food consumption. Previous studies have compared lg(Ba/Sr) values between different species of animals with differing diets, but few studies have been performed where animals of the same species consumed known diets ranging from completely terrestrial to completely marine. Additionally, how seaweed consumption affects dental and bone Ba, Sr and other trace element concentrations has not yet been directly investigated. In this study, tooth enamel from modern sheep (n = 15) that consumed known diets containing varying amounts of terrestrial grasses and seaweeds were analysed for their Sr, Ba, Ca, V, Mn, Co, Ni, As, and U concentrations. Additionally, δ13C values were analysed to enable comparison of δ13C and trace element ratios as markers of marine plant food consumption. The consumed vegetation types (grasses and seaweeds) were also analysed for trace element ratios, as were soils and sands from areas where the animals were pastured. To investigate how decay processes (i.e., diagenesis) may affect lg(Ba/Sr) in archaeological tooth enamel, teeth of 22 sheep from seven archaeological sites (ranging from ca. 5000 to 1000 years old) on the Orkney Islands, Scotland, were also analysed. The results show that tooth enamel from seaweed-eating sheep had significantly different lg(Ba/Sr) (−2.4 to −1.6) and δ13C values (−6.7‰ to −3.3‰) when compared to terrestrial-feeding sheep (lg(Ba/Sr) 0.6 to −0.5; δ13C −15.5‰ to −14.7‰), with a linear correlation between lg(Ba/Sr) and δ13C (R2 = 0.94). Vegetation, soil and sand results confirmed the assumed dependence of enamel lg(Ba/Sr) values on the (bioaccessible) Ba and Sr concentrations of the consumed matter. The archaeological samples had elevated relative amounts of U, V, As, Mn, Co, and Ni, attributable to diagenesis. However, the lg(Ba/Sr) values of the archaeological enamel followed the trend established using the modern samples, indicating that diagenesis did not cause significant changes in lg(Ba/Sr) in these samples. In conclusion, lg(Ba/Sr) values in enamel appear to be a useful indicator of the relative amount of marine food consumed, including seaweeds. This may be particularly advantageous for samples and locations where δ13C is unreliable or ambiguous as an indicator of marine food consumption

Xanthohumol attenuates tumour cell-mediated breaching of the lymphendothelial barrier and prevents intravasation and metastasis

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In vitro characterisation of the anti-intravasative properties of the marine product heteronemin

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Ageing and performance of warm mix asphalt pavements

This paper presents results from investigating the ageing behaviour and performance of different warm mix asphalt (WMA) pavement mixtures also called energy reduced pavements. The mixtures were either prepared in the laboratory or taken directly from a mixing plant. The study compared the rutting and fatigue behaviours of unaged material in comparison to long term laboratory aged material. In order to conduct the long term ageing, a special laboratory ageing protocol with different heating, cooling and watering cycles had been developed. The investigation revealed a quite controversial rutting behavior which could not be explained with the available data. While most aged energy reduced pavements showed increased rutting for other mixtures, lower rut depths could be found. As opposed to this finding, fatigue and stiffness of all aged energy reduced pavement samples compared to unaged samples improved significantly. The overall results led to the conclusion that the ageing of energy reduced pavement simulated in the laboratory is not very critical regarding their mechanical performance. Therefore, it was confirmed that the application of this type of pavement provides a good solution for saving on CO2 emissions. Another advantage is that by using energy reduced pavements the road construction season can be significantly prolonged

Automated Video-Based Analysis Framework for Behavior Monitoring of Individual Animals in Zoos Using Deep Learning - A Study on Polar Bears

The monitoring of animals under human care is a crucial tool for biologists and zookeepers to keep track of the animals’ physical and psychological health. Additionally, it enables the analysis of observed behavioral changes and helps to unravel underlying reasons. Enhancing our understanding of animals ensures and improves ex situ animal welfare as well as in situ conservation. However, traditional observation methods are time- and labor-intensive, as they require experts to observe the animals on-site during long and repeated sessions and manually score their behavior. Therefore, the development of automated observation systems would greatly benefit researchers and practitioners in this domain. We propose an automated framework for basic behavior monitoring of individual animals under human care. Raw video data are processed to continuously determine the position of the individuals within the enclosure. The trajectories describing their travel patterns are presented, along with fundamental analysis, through a graphical user interface (GUI). We evaluate the performance of the framework on captive polar bears (Ursus maritimus). We show that the framework can localize and identify individual polar bears with an F1 score of 86.4%. The localization accuracy of the framework is 19.9±7.6 cm, outperforming current manual observation methods. Furthermore, we provide a bounding-box-labeled dataset of the two polar bears housed in Nuremberg Zoo

Selection of scFv Antibody Fragments Binding to Human Blood versus Lymphatic Endothelial Surface Antigens by Direct Cell Phage Display

<div><p>The identification of marker molecules specific for blood and lymphatic endothelium may provide new diagnostic tools and identify new targets for therapy of immune, microvascular and cancerous diseases. Here, we used a phage display library expressing human randomized single-chain Fv (scFv) antibodies for direct panning against live cultures of blood (BECs) and lymphatic (LECs) endothelial cells in solution. After six panning rounds, out of 944 sequenced antibody clones, we retrieved 166 unique/diverse scFv fragments, as indicated by the V-region sequences. Specificities of these phage clone antibodies for respective compartments were individually tested by direct cell ELISA, indicating that mainly pan-endothelial cell (EC) binders had been selected, but also revealing a subset of BEC-specific scFv antibodies. The specific staining pattern was recapitulated by twelve phage-independently expressed scFv antibodies. Binding capacity to BECs and LECs and differential staining of BEC versus LEC by a subset of eight scFv antibodies was confirmed by immunofluorescence staining. As one antigen, CD146 was identified by immunoprecipitation with phage-independent scFv fragment. This antibody, B6-11, specifically bound to recombinant CD146, and to native CD146 expressed by BECs, melanoma cells and blood vessels. Further, binding capacity of B6-11 to CD146 was fully retained after fusion to a mouse Fc portion, which enabled eukaryotic cell expression. Beyond visualization and diagnosis, this antibody might be used as a functional tool. Overall, our approach provided a method to select antibodies specific for endothelial surface determinants in their native configuration. We successfully selected antibodies that bind to antigens expressed on the human endothelial cell surfaces <i>in situ</i>, showing that BECs and LECs share a majority of surface antigens, which is complemented by cell-type specific, unique markers.</p></div

Production of scFv-Fc B6-11 fusion antibody and reconfirmation of binding to CD146.

<p>A: Western Blot analysis showing molecular size of scFv-Fc fusion antibodies B6-11, B6-112 and B6-117. The scFv-region of the phagemids was cloned into the pFUSE-mIgG2B-vector (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#pone.0127169.s005" target="_blank">S5 Fig</a>). Constructs were transfected into HEK293-cells. Cell culture supernatants were loaded on reducing (R) and non-reducing (NR) 10% SDS-PAGE, and nitrocellulose blots were probed with peroxidase-labeled anti-mouseFc antibodies. scFv-Fc fusion antibodies are secreted as approximate 140 kDa dimers, as seen under non-reducing condition (-DTT). Control: Fc only protein, produced from pFUSE vector without scFv insert. B: Immunoprecipitation with scFv-Fc B6-11 reconfirms CD146-binding. G1S1 lysates were incubated with scFv-Fc B6-11 and Fc only as control. Blots of immunoprecipitates were probed with anti-CD146 and anti-mouseFc antibodies. C: scFv-Fc B6-11 binds to recombinant CD146 in ELISA. scFv-Fc B6-11, commercial anti-CD146 antibody and Fc only were used on respective dilutions of recombinant human CD146 or BSA as control antigen coated on 96-well ELISA plates. Absorbance was measured at 450nm. D: scFv-Fc B6-11 fusion antibody binds to cells expressing CD146 in ELISA. Purified scFv-Fc B6-11 (light grey bars), commercial anti-CD146 antibody (dark grey bars) and Fc only (black bars) were added to monolayers of respective cell lines. Bound antibodies were detected using anti-Fc and HRP-conjugated anti-rabbit antibodies, and absorbance was measured at 450nm. Mean and standard deviation of triplicate experiments are given. E: On HDMECs, scFv-Fc B6-11 fusion antibody shows the same reactivity as a commercial anti-CD146 antibody. F: scFv-Fc fusion antibodies (red) reveal diverse membraneous labling patterns in co-immunofluorescent stainings with anti-CD31 antibody (green). Nuclei were counterstained with DAPI. Size bars: 50μm.</p

Frequency of diverse scFv antibody sequences among 557 intact clones.

<p>Out of 994 sequenced phage clones, 557 intact scFv sequences were derived, among which 166 diverse scFv sequences were identified (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#pone.0127169.s012" target="_blank">S2 Table</a>).</p><p>^a Frequency of unique scFv antibody sequences</p><p>^b Number of different scFv sequences</p><p>^c Number of scFv sequences with respective sequence diversity</p><p>^d Sequence occurrence was calculated as percentage of sequence count.</p><p>Frequency of diverse scFv antibody sequences among 557 intact clones.</p

LC-MS/MS identification of CD146 binding to scFv B6-11, and antigen confirmation by immunoprecipitation and ELISA.

<p>A: Alignment of LC-MS/MS identified peptides (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#pone.0127169.s004" target="_blank">S4 Fig</a>) with the sequence of MCAM/CD146MUC18. The eluates from scFv B6-11 immunoprecipitation were subjected to trypsin digestion (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#sec002" target="_blank">Materials and Methods</a> section) and subsequently analyzed by LC-MS/MS. B: Immunoprecipitation of CD146 by soluble scFv B6-11 from BEC lysates. Immune complexes were tested by Western blot in reducing conditions using commercial anti-CD146 antibody. Lane 1: input BEC lysate, lanes 2–5: immunoprecipitates with scFv B6-11, lanes 2 and 4: under addition of PNGase F. Treatment of BEC lysates with PNGase prior or after addition of scFv B6-11 had no influence on co-immunoprecipitation capacity of scFv B6-11, showing that scFv B6-11 binding to CD146 is glycosylation-independent. C: scFv B6-11 binds to immobilized extracellular domain of recombinant human CD146 in ELISA. An irrelevant antigen (BSA), a non-binding scFv and uncoated wells served as controls. scFv binding was detected with peroxidase-conjugated anti-His tag antibody (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#pone.0127169.s011" target="_blank">S1 Table</a>), and absorbance was measured at 450nm. Mean and standard deviation of triplicate experiments are given. D: CD146 expression of different cell lines as shown by immunoprobing with anti-CD146 antibody. CD146 is expressed in BECs, in A375, CRL1676, HTB71 melanoma cells, but not in primary LECs and HEK293 cells. The same blot was probed with anti-tubulin antibody for control of equal protein loads. E: scFv B6-11 stains cell lines expressing CD146 with similar intensity as commercial anti-CD146 antibody in ELISA. Negative controls were a non-binding scFv and 2^nd antibody only. F: Similar to commercial anti-CD146 antibody, scFv B6-11 stains BECs (upper lane, red) but not LECs (lower lane, green) in immunofluorescence. Size bars: 50μm. G: scFv B6-11 stains blood, but not lymphatic vessels in human skin. Double immunofluorescence staining of skin sample with Cy3-labeled scFv-B6-11 or anti-CD146 (red) and anti-PDPN (green) antibodies. Blood (BV) and lymphatic (LV) vessels are indicated by lines. Nuclei were counterstained with DAPI. Size bars: 50μm.</p