Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor

FFA2 is a G protein-coupled receptor that responds to short chain fatty acids (SCFAs) and has generated interest as a therapeutic target for metabolic and inflammatory conditions. However, definition of its functions has been slowed by a dearth of selective ligands that can distinguish it from the closely related FFA3. At present, the only selective ligands described for FFA2 suffer from either poor potency, altered signaling due to allosteric modes of action, or a lack of function at non-human orthologs of the receptor. To address the need for novel selective ligands, we synthesized two compounds potentially having FFA2 activity and examined the molecular basis of their function. These compounds were confirmed to be potent and selective FFA2 agonists that interact with the orthosteric binding site. A combination of ligand structure-activity relationship, pharmacological analysis, homology modeling, species ortholog comparisons and mutagenesis studies were then employed to define the molecular basis of selectivity and function of these ligands. From this, we identified key residues within both extracellular loop 2 (ECL2) and the transmembrane domain (TM) regions of FFA2 critical for ligand function. One of these ligands was active with reasonable potency at rodent orthologs of FFA2 and demonstrated the role of FFA2 in the regulation of lipolysis in murine 3T3-L1 adipocytes. Together, these findings describe the first potent and selective FFA2 orthosteric agonists and demonstrate key aspects of ligand interaction within the orthosteric binding site of FFA2 that will be invaluable in future ligand development at this receptor.

Arabidopsis Rab-E GTPases exhibit a novel interaction with a plasma-membrane phosphatidylinositol-4-phosphate 5-kinase

Rab GTPases of the Arabidopsis Rab-E subclass are related to mammalian Rab8 and are implicated in membrane trafficking from the Golgi to the plasma membrane. Using a yeast two-hybrid assay, Arabidopsis phosphatidylinositol-4-phosphate 5-kinase 2 (PtdIns(4)P 5-kinase 2; also known as PIP5K2), was shown to interact with all five members of the Rab-E subclass but not with other Rab subclasses residing at the Golgi or trans-Golgi network. Interactions in yeast and in vitro were strongest with RAB-E1d[Q74L] and weakest with the RAB-E1d[S29N] suggesting that PIP5K2 interacts with the GTP-bound form. PIP5K2 exhibited kinase activity towards phosphatidylinositol phosphates with a free 5-hydroxyl group, consistent with PtdIns(4)P 5-kinase activity and this activity was stimulated by Rab binding. Rab-E proteins interacted with PIP5K2 via its membrane occupancy and recognition nexus (MORN) domain which is missing from animal and fungal PtdIns(4)P 5-kinases. In plant cells, GFP:PIP5K2 accumulated at the plasma membrane and caused YFP:RAB-E1d to relocate there from its usual position at the Golgi. GFP:PIP5K2 was rapidly turned over by proteasomal activity in planta, and overexpression of YFP:PIP5K2 caused pleiotropic growth abnormalities in transgenic Arabidopsis. We propose that plant cells exhibit a novel interaction in which PIP5K2 binds GTP-bound Rab-E proteins, which may stimulate temporally or spatially localized PtdIns(4,5)P(2) production at the plasma membrane.

Metamaterials-Based Label-Free Nanosensor for Conformation and Affinity Biosensing

Analysis of molecular interaction and conformational dynamics of biomolecules is of paramount importance in understanding of their vital functions in complex biological systems, disease detection, and new drug development. Plasmonic biosensors based upon surface plasmon resonance and localized surface plasmon resonance have become the predominant workhorse for detecting accumulated biomass caused by molecular binding events. However, unlike surface-enhanced Raman spectroscopy (SERS), the plasmonic biosensors indeed are not suitable tools to interrogate vibrational signatures of conformational transitions required for biomolecules to interact. Here, we show that highly tunable plasmonic metamaterials can offer two transducing channels for parallel acquisition of optical transmission and sensitive SERS spectra at the biointerface, simultaneously probing the conformational states and binding affinity of biomolecules, e.g. G-quadruplexes, in different environments. We further demonstrate the use of the metamaterials for fingerprinting and detection of arginine-glycine-glycine domain of nucleolin, a cancer biomarker which specifically binds to a G-quadruplex, with the picomolar sensitivity.

Learner-Teacher Interaction (LTI): engaging and supporting students in achieving self-regulated learning

This report represents the second stage of a study which was part of a wide-ranging research programme conducted by the Centre for Excellence of Interprofessional Education, Queen’s University Belfast. The study was an investigation into learner-teacher interaction in the education of undergraduate medical and other healthcare students in order to inform how teachers might facilitate learning in a healthcare setting. It focused in particular on clinical and ward-based tutorials and seminars.

In order to give meaning to this second stage of the study, the report will contextualise the learner-teacher interaction study, will describe the research methods and methods of analysis developed and used to explore learner-teacher interaction. It will then focus on this second stage of the research and the results of the analysis of video sessions of clinical and ward-based tutorials and seminars. In particular it will identify examples of good practice and missed opportunities for the engagement of the learners.

Living in The Matrix: Virtual Reality Systems and Hyperspatial Representation in Architecture

In the digital age, the hyperspace of virtual reality systems stands out as a new spatial concept creating a parallel realm to "real" space. Virtual reality influences one’s experience of and interaction with architectural space. This "otherworld" brings up the criticism of the existing conception of space, time and body. Hyperspaces are relatively new to designers but not to filmmakers. Their cinematic representations help the comprehension of the outcomes of these new spaces. Visualisation of futuristic ideas on the big screen turns film into a medium for spatial experimentation. Creating a possible future, The Matrix (Andy and Larry Wachowski, 1999) takes the concept of hyperspace to a level not-yet-realised but imagined. With a critical gaze at the existing norms of architecture, the film creates new horizons in terms of space. In this context, this study introduces science fiction cinema as a discussion medium to understand the potentials of virtual reality systems for the architecture of the twenty first century. As a "role model" cinema helps to better understand technological and spatial shifts. It acts as a vehicle for going beyond the spatial theories and designs of the twentieth century, and defining the conception of space in contemporary architecture.

Asymmetric Total Synthesis of (+)-Inthomycin C Via O-Directed Free Radical Alkyne Hydrostannation with Ph3SnH and Catalytic Et3B: Reinstatement of the Zeeck-Taylor (3R)-Structure for (+)-Inthomycin C

A new pathway to (+)-inthomycin C is reported that exploits an O-directed free radical hydrostannation reaction on (−)-12 and a Stille cross-coupling as key steps. Significantly, the latter process was effected on 19 where a gauche-pentane repulsive interaction could interfere. Our stereochemical studies on the alkynol (−)-12 and the enyne (+)-7 confirm that Ryu and Hatakeyama’s (3S)-stereochemical revision of (+)-inthomycin C is invalid and that Zeeck and Taylor’s original (3R)-stereostructure for (+)-inthomycin C is correct.

Wearable inkjet-printed antenna performance for medical applications at 868/915 MHz

The use of biosensors attached to the body for health monitoring is now readily accepted, and the merits of such systems and their potential impact on healthcare receive much attention. Wearable medical systems used in clinical applications to monitor vital signs must be comfortable to wear, yet have robust performance to ensure reliable communications links. Additionally, and vital to the success of these innovations, is that these solutions are disposable to avoid risk of patient infection and this means that they must be ultra-low cost. Antennas optimized for printing using conductive inks offer new exciting advances in making a truly disposable solution.

Do mirrors reflect reality in agonistic encounters? A test of mutual cooperation in displays

Animals frequently engage in mutual displays that may allow or at least help decisions about the outcome of agonistic encounters with mutual benefit to the opponents. In fish these often involve lateral displays, with previous studies finding evidence of population-level lateralization with a marked preference for showing the right side and using the right eye. Because both opponents tend to show this preference a head to tail configuration is formed and is used extensively during the display phase. Here we tested the significance of these lateral displays by comparing displays to a mirror with those to a real opponent behind a transparent barrier. The frequency of displays was lower to a mirror but the individual displays were of greater duration indicating a slower pace of the interaction with a mirror. This suggests that fish respond to initiatives of real opponents but as mirror images do not initiate moves the focal fish only moves when it is ready to change position. However, lateralization was still found with mirrors, indicating that the right-side bias is a feature of the individual and not of the interaction between opponents. We discuss implications for ideas about the evolution of mutual cooperation and information exchange in contests, as well as the utility of the use of mirrors in the study of aggression in fish.

Ultra-broadband Polarisers Based on Metastable Free-Standing Aligned Carbon Nanotube Membranes

A carbon nanotube free-standing linearly dichroic polariser is developed using solid-state extrusion. Membrane cohesion is experimentally and numerically demonstrated to derive from inter-tube van der Waals interactions in this family of planar metastable morphologies, controlled by the chemical vapour deposition conditions. Ultra-broadband polarisation (400 nm – 2.5 mm) is shown and corroborated by effective medium and full numerical simulations.

Density functional theory study of the interaction between CO and on a Pt surface:CO/Pt(111), O/Pt(111), and CO/O/Pt(111)

Ab initio total energy calculations within the Density Functional Theory framework were carried out for Pt(111), Pt(111)-p(2x2)-CO, Pt(111)-p(2x2)-O, and Pt(111)-p(2x2)-(CO+O) to provide an insight into the interaction between CO and O on metal surfaces, an important issue in CO oxidation, and also in promotion and poisoning effects of catalysis. The geometrical structures of these systems were optimized with respect to the total energy, the results of which agree with existing experimental values very well. It is found that (i) the local structures of Pt(111)-p(2x2)-(CO+O), such as the bond lengths of C-O, C-Pt, and O-Pt (chemisorbed O atom with Pt), are almost the same as that in Pt(111)-p(2x2)-CO and Pt(111)-p(2x2)-O, respectively, (ii) the total valence charge density distributions in Pt(111)-p(2x2)-(CO+O) are very similar to that in Pt(111)-p(2x2)-CO, except in the region of the chemisorbed oxygen atom, and also nearly identical to that in Pt(111)-p(2x2)-O, apart from in the region of the chemisorbed CO, and (iii) the chemisorption energy of CO on a precovered Pt(111)-p(2x2)-O and the chemisorption energy of O on a precovered Pt(111)-p(2x2)CO are almost equal to that in Pt(111)-p(2x2)-CO and Pt(111)-p(2x2)-O, respectively. These results indicate that the interaction between CO and chemisorbed oxygen on a metal surface is mainly shore range in nature. The discussions of Pt-CO and Pt-O bonding and the interaction between CO and the chemisorbed oxygen atom on Pt(111) are augmented by local densities of states and real space distributions of quantum states.

Maintaining a grasp on reality – Is our validation and design effort focused in the same direction?

This article examines the influence on the engineering design process of the primary objective of validation, whether it is proving a model, a technology or a product. Through the examination of a number of stiffened panel case studies, the relationships between simulation, validation, design and the final product are established and discussed. The work demonstrates the complex interactions between the original (or anticipated) design model, the analysis model, the validation activities and the product in service. The outcome shows clearly some unintended consequences. High fidelity validation test simulations require a different set of detailed parameters to accurately capture behaviour. By doing so, there is a divergence from the original computer-aided design model, intrinsically limiting the value of the validation with respect to the product. This work represents a shift from the traditional perspective of encapsulating and controlling errors between simulation and experimental test to consideration of the wider design-test process. Specifically, it is a reflection on the implications of how models are built and validated, and the effect on results and understanding of structural behaviour. This article then identifies key checkpoints in the design process and how these should be used to update the computer-aided design system parameters for a design. This work strikes at a fundamental challenge in understanding the interaction between design, certification and operation of any complex system.

Understanding the Interaction between Low-Energy Electrons and DNA Nucleotides in Aqueous Solution

Reactions that can damage DNA have been simulated using a combination of molecular dynamics and density functional theory. In particular, the damage caused by the attachment of a low energy electron to the nucleobase. Simulations of anionic single nucleotides of DNA in an aqueous environment that was modeled explicitly have been performed. This has allowed us to examine the role played by the water molecules that surround the DNA in radiation damage mechanisms. Our simulations show that hydrogen bonding and protonation of the nucleotide by the water can have a significant effect on the barriers to strand breaking reactions. Furthermore, these effects are not the same for all four of the bases.

Incorporation of spiritual care as a component of healthcare and medical education: comparison of viewpoints of healthcare providers from a rural community hospital in Uganda with those of medical students from United Kingdrom

This study addresses cultural differences regarding views on the place for spirituality within healthcare training and delivery. A questionnaire was devised using a 5-point ordinal scale, with additional free text comments assessed by thematic analysis, to compare the views of Ugandan healthcare staff and students with those of (1) visiting international colleagues at the same hospital; (2) medical faculty and students in United Kingdom. Ugandan healthcare personnel were more favourably disposed towards addressing spiritual issues, their incorporation within compulsory healthcare training, and were more willing to contribute themselves to delivery than their European counterparts. Those from a nursing background also attached a greater importance to spiritual health and provision of spiritual care than their medical colleagues. Although those from a medical background recognised that a patient’s religiosity and spirituality can affect their response to their diagnosis and prognosis, they were more reticent to become directly involved in provision of such care, preferring to delegate this to others with greater expertise. Thus, differences in background, culture and healthcare organisation are important, and indicate that the wide range of views expressed in the current literature, the majority of which has originated in North America, are not necessarily transferable between locations; assessment of these issues locally may be the best way to plan such training and incorporation of spiritual care into clinical practice.

Label-free based quantitative proteomics analysis of primary neonatal porcine Leydig cells exposed to the persistent contaminant 3-methylsulfonyl-DDE

Evidence that persistent environmental pollutants may target the male reproductive system is increasing. The male reproductive system is regulated by secretion of testosterone by testicular Leydig cells, and perturbation of Leydig cell function may have ultimate consequences. 3-Methylsulfonyl-DDE (3-MeSO₂-DDE) is a potent adrenal toxicants formed from the persistent insecticide DDT. Although studies have revealed the endocrine disruptive effect of 3-MeSO₂-DDE, the underlying mechanisms at cellular level in steroidogenic Leydig cells remains to be established. The current study addresses the effect of 3-MeSO₂-DDE on viability, hormone production and proteome response of primary neonatal porcine Leydig cells. The AlamarBlue™ assay was used to evaluate cell viability. Solid phase radioimmunoassay was used to measure concentration of hormones produced by both unstimulated and Luteinizing hormone (LH)-stimulated Leydig cells following 48h exposure. Protein samples from Leydig cells exposed to a non-cytotoxic concentration of 3-MeSO₂-DDE (10μM) were subjected to nano-LC-MS/MS and analyzed on a Q Exactive mass spectrometer and quantified using label-free quantitative algorithm. Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) were carried out for functional annotation and identification of protein interaction networks. 3-MeSO₂-DDE regulated Leydig cell steroidogenesis differentially depending on cell culture condition. Whereas its effect on testosterone secretion at basal condition was stimulatory, the effect on LH-stimulated cells was inhibitory. From triplicate experiments, a total of 6804 proteins were identified in which the abundance of 86 proteins in unstimulated Leydig cells and 145 proteins in LH-stimulated Leydig cells was found to be significantly regulated in response to 3-MeSO₂-DDE exposure. These proteins not only are the first reported in relation to 3-MeSO₂-DDE exposure, but also display small number of proteins shared between culture conditions, suggesting the action of 3-MeSO₂-DDE on several targeted pathways, including mitochondrial dysfunction, oxidative phosphorylation, EIF2-signaling, and glutathione-mediated detoxification. Further identification and characterization of these proteins and pathways may build our understanding to the molecular basis of 3-MeSO₂-DDE induced endocrine disruption in Leydig cells.

Free-roaming dogs control activities in one Italian province (2000-2013): Is the implemented approach effective?

In Italy, standards for the management of free-roaming dogs (FRDs) are defined by regional norms, generating a high variability of approaches around the country. Despite efforts carried out by the competent authorities, FRDs are still a reality impacting upon animal health and welfare and public costs. A similar scenario can be found in many other Mediterranean and Balkan counties. Here we present 14 years of data (2000–2013) retrieved from the admission dog registry of a public shelter (PS) responsible for the collection of stray dogs from one Italian province. The aim of this retrospective study was to describe the local FRD population, identifying its source and to evaluate the effectiveness of the actions implemented by the local authorities. In the investigated period, 7,475 dogs were admitted to the PS. Despite the intense sterilisation plan (mean 381.7 sterilisations per year), the overall number of dogs entering PS did not decrease consistently across the years. Results highlighted a lack of responsibility of owners by failing to sterilise and identify their dogs and allowing intact animals to roam free, therefore producing uncontrolled and unwanted litters. The current dog population management strategy, based on both sheltering and capture-neuter-release programmes, is insufficient to tackle the straying phenomenon. Educational and sterilisation programmes should be an integral part of a successfully implemented FRD control plan. Our results provide further insight on free-roaming dog population dynamics and control systems, and may have important implications for many other local contexts across Europe trying to overcome the straying phenomenon.