Deletion of the Androgen Receptor in Adipose Tissue in Male Mice Elevates Retinol Binding Protein 4 and Reveals Independent Effects on Visceral Fat Mass and on Glucose Homeostasis

Testosterone deficiency is epidemic in obese ageing males with type 2 diabetes, but the direction of causality remains unclear. Testosterone-deficient males and global androgen receptor (AR) knockout mice are insulin resistant with increased fat, but it is unclear whether AR signaling in adipose tissue mediates body fat redistribution and alters glucose homoeostasis. To investigate this, mice with selective knockdown of AR in adipocytes (fARKO) were generated. Male fARKO mice on normal diet had reduced perigonadal fat but were hyperinsulinemic and by age 12 months, were insulin deficient in the absence of obesity. On high-fat diet, fARKO mice had impaired compensatory insulin secretion and hyperglycemia, with increased susceptibility to visceral obesity. Adipokine screening in fARKO mice revealed a selective increase in plasma and intra-adipose retinol binding protein 4 (RBP4) that preceded obesity. AR activation in murine 3T3 adipocytes downregulated RBP4 mRNA. We conclude that AR signaling in adipocytes not only protects against high-fat diet–induced visceral obesity but also regulates insulin action and glucose homeostasis, independently of adiposity. Androgen deficiency in adipocytes in mice resembles human type 2 diabetes, with early insulin resistance and evolving insulin deficiency

Using Green Emitting pH-Responsive Nanogels to Report Environmental Changes within Hydrogels: A Nanoprobe for Versatile Sensing

Remotely reporting the local environment within hydrogels using inexpensive laboratory techniques has excellent potential to improve our understanding of the nanometer-scale changes that cause macroscopic swelling or deswelling. Whilst photoluminescence (PL) spectroscopy is a popular method for such studies this approach commonly requires bespoke and time-consuming synthesis to attach fluorophores which may leave toxic residues. A promising and more versatile alternative is to use a pre-formed nanogel probe that contains a donor/acceptor pair and then “dope” that into the gel during gel assembly. Here, we introduce green-emitting methacrylic acid-based nanogel probe particles and use them to report the local environment within four different gels as well as stem cells. As the swelling of the nanogel probe changes within the gels the non-radiative energy transfer efficiency is strongly altered. This efficiency change is sensitively reported using the PL ratiometric intensity from the donor and acceptor. We demonstrate that our new nanoprobes can reversibly report gel swelling changes due to five different environmental stimuli. The latter are divalent cations, gel degradation, pH changes, temperature changes and tensile strain. In the latter case, the nanoprobe rendered a nanocomposite gel mechanochromic. The results not only provide new structural insights for hierarchical natural and synthetic gels, but also demonstrate that our new green-fluorescing nanoprobes provide a viable alternative to custom fluorophore labelling for reporting the internal gel environment and its changes

Thermoresponsive magnetic colloidal gels via surface-initiated polymerisation from functional microparticles

Novel magnetothermally responsive core-shell microparticles have been synthesized. The aqueous suspensions of these particles exhibit fast thermoreversible fluid-to-gel transitions and retain good magnetic properties. Rheological measurements demonstrated that the viscoelasticity of the prepared particle gels can be tuned, enabling these gels to have the mechanical properties that should facilitate their applications as 3D cell scaffolds for in vitro expansion of cells. Also, it was found that the responsive particles could be used in repeated heating-cooling cycles without marked changes in gel elasticity. Presto Blue viability assays of 3T3 fibroblasts and human mesenchymal sem cells cultured within the colloidal gel showed that the cells remained viable and proliferated, with significant increases in cell numbers over extended culture times. Confocal microscopy images of 3T3 cells cultured within the colloidal gel demonstrated that cells adhered, spread and retained their normal morphologies during proliferation.. Furthermore, magnetic separation allowed efficient recovery of cells after their expansion in vitro without need for enzyme-mediated release steps. Trypsin-free cell passages were performed allowing multiple growth, separation and reloading of cells within the colloidal gels. Overall, the results suggest this colloidal gel has potential as a 3D scaffold for in vitro expansion of a variety of cell types and for enzyme free cell harvesting

Highly Swelling pH-Responsive Microgels for Dual Mode Near Infra-Red Fluorescence Reporting and Imaging

Near infra-red (NIR) fluorescence is a desirable property for probe particles because such deeply penetrating light enables remote reporting of the local environment in complex surroundings and imaging. Here, two NIR non-radiative energy transfer (NRET) fluorophores (Cy5 and Cy5.5) are coupled to preformed pH-responsive poly(ethylacrylate-methacrylic acid-divinylbenzene) microgel particles (PEA-MAA-5/5.5 MGs) to obtain new NIR fluorescent probes that are cytocompatible and swell strongly. NIR ratiometric photoluminescence (PL) intensity analysis enables reporting of pH-triggered PEA-MAA-5/5.5 MG particle swelling ratios over a very wide range (from 1–90). The dispersions have greatly improved colloidal stability compared to a reference temperature-responsive NIR MG based on poly(N-isopropylacrylamide) (PNP-5/5.5). We also show that the wavelength of maximum PL intensity (λmax) is a second PL parameter that enables remote reporting of swelling for both PEA-MAA-5/5.5 and PNP-5/5.5 MGs. After internalization the PEA-MAA-5/5.5 MGs are successfully imaged in stem cells using NIR light. They are also imaged after subcutaneous injection into model tissue using NIR light. The new NIR PEA-MAA-5/5.5 MGs have excellent potential for reporting their swelling states (and any changes) within physiological settings as well as very high ionic strength environments (e.g., waste water)

Trace-element abundances in the shallow lithospheric mantle of the North Atlantic Craton margin: implications for melting and metasomatism beneath Northern Scotland

Bulk rock geochemistry and major- and trace-element compositions of clinopyroxene have been determined for three suites of peridotitic mantle xenoliths from the North Atlantic Craton (NAC) in northern Scotland, to establish the magmatic and metasomatic history of subcontinental lithospheric mantle (SCLM) below this region. Spinel lherzolites from the southernmost locality (Streap Com'laidh) have non-NAC mantle compositions, while the two northern xenolith suites (Loch Roag and Rinibar) are derived from the thinned NAC marginal keel. Clinopyroxene compositions have characteristic trace-element signatures which show both 'primary' and 'metasomatic' origins. We use Zr and Hf abundances to identify ancient cryptic refertilization in 'primary' clinopyroxenes. We suggest that Loch Roag and Rinibar peridotite xenoliths represent an ancient Archaean-Palaeoproterozoic SCLM with original depleted cratonic signatures which were overprinted by metasomatism around the time of intrusion of the Scourie Dyke Swarm (∼2.4 Ga). This SCLM keel was preserved during Caledonian orogenesis, although some addition of material and/or metasomatism probably also occurred, as recorded by Rinibar xenoliths. Rinibar and Streap xenoliths were entrained in Permo-Carboniferous magmas and thus were isolated from the SCLM ∼200 Ma before Loch Roag xenoliths (in an Eocene dyke). Crucially, despite their geographical location, lithospheric mantle peridotite samples from Loch Roag show no evidence of recent melting or refertilization during the Palaeogene opening of the Atlantic

A thermoresponsive and magnetic colloid for 3D cell expansion and reconfiguration

A dual thermoresponsive and magnetic colloidal gel matrix is described for enhanced stem-cell culture. The combined properties of the material allow enzyme-free passaging and expansion of mesenchymal stem cells, as well as isolation of cells postculture by the simple process of lowering the temperature and applying an external magnetic field. The colloidal gel can be reconfigured with thermal and magnetic stimuli to allow patterning of cells in discrete zones and to control movement of cells within the porous matrix during culture

The Alliance for Cellular Signaling Plasmid Collection: A Flexible Resource for Protein Localization Studies and Signaling Pathway Analysis

Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks. Analysis of these relationships requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling, we developed a robust method for cloning large numbers of signaling ORFs into Gateway® entry vectors, and we created a wide range of compatible expression platforms for proteomics applications. To date, we have generated over 3000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at www.signaling-gateway.org/data/plasmid/ that allows users to browse, search, and blast Alliance for Cellular Signaling plasmids. The collection primarily contains murine signaling ORFs with an emphasis on kinases and G protein signaling genes. Here we describe the cloning, databasing, and application of this proteomics resource for large scale subcellular localization screens in mammalian cell lines

EXACT2: the semantics of biomedical protocols

© 2014 Soldatova et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.This article has been made available through the Brunel Open Access Publishing Fund.Background: The reliability and reproducibility of experimental procedures is a cornerstone of scientific practice. There is a pressing technological need for the better representation of biomedical protocols to enable other agents (human or machine) to better reproduce results. A framework that ensures that all information required for the replication of experimental protocols is essential to achieve reproducibility. Methods: We have developed the ontology EXACT2 (EXperimental ACTions) that is designed to capture the full semantics of biomedical protocols required for their reproducibility. To construct EXACT2 we manually inspected hundreds of published and commercial biomedical protocols from several areas of biomedicine. After establishing a clear pattern for extracting the required information we utilized text-mining tools to translate the protocols into a machine amenable format. We have verified the utility of EXACT2 through the successful processing of previously ‘unseen’ (not used for the construction of EXACT2) protocols. Results: The paper reports on a fundamentally new version EXACT2 that supports the semantically-defined representation of biomedical protocols. The ability of EXACT2 to capture the semantics of biomedical procedures was verified through a text mining use case. In this EXACT2 is used as a reference model for text mining tools to identify terms pertinent to experimental actions, and their properties, in biomedical protocols expressed in natural language. An EXACT2-based framework for the translation of biomedical protocols to a machine amenable format is proposed. Conclusions: The EXACT2 ontology is sufficient to record, in a machine processable form, the essential information about biomedical protocols. EXACT2 defines explicit semantics of experimental actions, and can be used by various computer applications. It can serve as a reference model for for the translation of biomedical protocols in natural language into a semantically-defined format.This work has been partially funded by the Brunel University BRIEF award and a grant from Occams Resources