Database mining studies on protein-peptide and protein-protein interactions

A major area of interest is the identification of proteins that play a role in hormone dependent cancers and in collaboration with the MRC Centre for Reproductive Health we studied the gonadotropin releasing hormone receptor (GnRH-R). Other targets described in the thesis are the SH3 domain of PSD-95 and the protein BLyS. In order to identify potential inhibitory small molecules we have used a variety of computational data base mining approaches as well as using and developing experimental binding assays. It has become increasingly challenging to evaluate the most representative drug like small molecule compounds when using traditional high throughput screening methods. This thesis assesses the use of in silico tools to probe key protein-protein and protein-peptide interactions. These tools provide a means to identify enriched compound datasets which can be purchased and tested in vitro in a time and cost efficient way. The transmembrane protein GnRH-R provides an interesting opportunity to identify small molecules that could inhibit the binding of its peptide ligand GnRH. This is a challenging project as there are few examples in the literature of drug-like molecules that bind to such protein-peptide interfaces. The first step involved receptor modelling using solved crystal structures of homologous proteins. The model was then validated by developing structure activity relationships for established high affinity ligands. We also performed crystallographic and biophysical studies on the native GnRH decapeptide. Two other protein-protein systems were also examined using the same virtual screening and experimental ligand binding methodology. SH3 domains play an important role in cell signalling and we used the PSD-95 protein as our target for study as a crystal structure has been published. As well as identifying potential ligands we characterised structural properties of PSD-95 fusion proteins and also developed the basis for compound assay. The third system studied was B Lymphocyte Stimulator (BLyS) which is a target for treatment of a number of autoimmune diseases. This presented an interesting target for study as the protein binds to multiple receptors depending on its multimeric state. BLyS protein was characterised using electron microscopy and other biophysical techniques

Biosynthesis of selenate reductase in <i>Salmonella enterica</i>:critical roles for the signal peptide and DmsD

Salmonella enterica serovar Typhimurium is a Gram-negative bacterium with a flexible respiratory capability. Under anaerobic conditions, S. enterica can utilize a range of terminal electron acceptors, including selenate, to sustain respiratory electron transport. The S. enterica selenate reductase is a membrane-bound enzyme encoded by the ynfEFGH-dmsD operon. The active enzyme is predicted to comprise at least three subunits where YnfE is a molybdenum-containing catalytic subunit. The YnfE protein is synthesized with an N-terminal twin-arginine signal peptide and biosynthesis of the enzyme is coordinated by a signal peptide binding chaperone called DmsD. In this work, the interaction between S. enterica DmsD and the YnfE signal peptide has been studied by chemical crosslinking. These experiments were complemented by genetic approaches, which identified the DmsD binding epitope within the YnfE signal peptide. YnfE signal peptide residues L24 and A28 were shown to be important for assembly of an active selenate reductase. Conversely, a random genetic screen identified the DmsD V16 residue as being important for signal peptide recognition and selenate reductase assembly

Abundance, efficiency, and stability of reference transcript expression in a seasonal rodent: The Siberian hamster

Quantitative PCR (qPCR) is a common molecular tool to analyse the expression of transcripts in non-traditional animal models. Most animals experience tissue-specific seasonal changes in cell structure, growth, and cellular function. As a consequence, the choice of reference or ‘house-keeping’ genes is essential to standardize expression levels of target transcripts of interest for qPCR analyses. This study aimed to determine the abundance, efficiency and stability of several reference genes commonly used for normalisation of qPCR analyses in a model of seasonal biology: the Siberian hamster (Phodopus sungorus). Liver, brown-adipose tissue (BAT), white adipose tissue (WAT), testes, spleen, kidney, the hypothalamic arcuate nucleus, and the pituitary gland from either long or short photoperiod Siberian hamsters were dissected to test tissue-specific and photoperiod effects on reference transcripts. qPCR was conducted for common reference genes including 18s ribosomal RNA (18s), glyceraldehyde 3-phosphate dehydrogenase (Gapdh), hypoxanthine-guanine phosphoribosyltransferase (Hprt), and actin-β (Act). Cycling time (Ct), efficiency (E) and replicate variation of Ct and E measured by percent coefficient of variance (CV%) was determined using PCR miner. Measures of stability were assessed using a combined approach of NormFinder and BestKeeper. 18s and Act did not vary in Ct across photoperiod conditions. Splenic, WAT and BAT Gapdh Ct was higher in long compared to short photoperiod. Splenic Hprt Ct was higher in long photoperiods. There was no significant effect of photoperiod, tissue or interaction on measures of efficiency, Ct CV%, or efficiency CV%. NormFinder and BestKeeper confirmed that 18s, Gapdh and Hprt were highly stable, while Act showed low stability. These findings suggest that 18s and Hprt show the most reliable stability, efficiency, and abundance across the tissues. Overall, the study provides a comprehensive and standardised approach to assess multiple reference genes in the Siberian hamster and help to inform molecular assays used in studies of photoperiodism

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

Transcriptome analyses of nine endocrine tissues identifies organism-wide transcript distribution and structure in the Siberian hamster

Temperate zone animals exhibit seasonal variation in multiple endocrine systems. In most cases, peripheral organs display robust switches in tissue involution and recrudescence in mass. Our understanding of the molecular control of tissue-specific changes in seasonal function remains limited. Central to this problem is the lack of information on the nucleic acid structure, and distribution of transcripts across tissues in seasonal model organisms. Here we report the transcriptome profile of nine endocrine tissues from Siberian hamsters. Luteinizing hormone receptor expression was localized to gonadal tissues and confirmed previous distribution analyses. Assessment of the prolactin receptor reveal relatively high abundance across tissues involved in reproduction, energy, and water homeostasis. Neither melatonin receptor-1a, nor -1b, were found to be expressed in most tissues. Instead, the closely related G-protein coupled receptor Gpr50 was widely expressed in peripheral tissues. Epigenetic enzymes such as DNA methyltransferase 3a, was widely expressed and the predominant DNA methylation enzyme. Quantitative PCR analyses revealed some sex- and tissue-specific differences for prolactin receptor and DNA methyltransferase 3a expression. These data provide significant information on the distribution of transcripts, relative expression levels and nucleic acid sequences that will facilitate molecular studies into the seasonal programs in mammalian physiology

Primer sequences and qPCR parameters.

Primer sequences and qPCR parameters.</p

Neural programming of seasonal physiology in birds and mammals: a modular perspective

Most animals in the temperate zone exhibit robust seasonal rhythms in neuroendocrine, physiological and behavioral processes. The integration of predictive and supplementary environmental cues (e.g., nutrients) involves a series of discrete, and interconnected brain regions that span hypothalamic, thalamic, mesencephalic, and limbic regions. Species-specific adaptive changes in these neuroendocrine structures and cellular plasticity have likely evolved to support seasonal life-history transitions. Despite significant advances in our understanding of ecological responses to predictive and supplementary environmental cues, there remains a paucity of literature on how these diverse cues impact the underlying neural and cellular substrates. To date, most scientific approach has focused on neuroendocrine responses to annual changes in daylength, referred to as photoperiod, due to the robust physiological changes to light manipulations in laboratory settings. In this review, we highlight the relatively few animal models that have been effectively used to investigate how predictive day lengths, and supplementary cues are integrated across hypothalamic nuclei, and discuss key findings of how seasonal rhythms in physiology are governed by adaptive neuroendocrine changes. We discuss how specific brain regions integrate environmental cues to form a complex multiunit or ‘modular’ system that has evolved to optimize the timing of seasonal physiology. Overall, the review aims to highlight the existence of a modular network of neural regions that independently contribute to timing seasonal physiology. This paper proposes that a multi-modular neuroendocrine system has evolved in which independent neural ‘units’ operate to support species-specific seasonal rhythms

FSHβ links photoperiodic signalling to seasonal reproduction in Japanese quail

Endogenous programs in avian reproductive seasonal life history transitions have been known for over 30 years. Attempts to identify the neural and molecular substrates of photoperiodic time measurement have, to date, focussed on blunt changes in light exposure during a restricted period of photoinducibility. Here we report mediobasal hypothalamus (MBH) and pituitary gland specific transcript expression cascades associated with photoperiod-induced seasonal life history transitions. We identified multiple waves of transcript expression that were not synchronized across brain regions. Follicle-stimulating hormone-β (FSHβ) expression increased during the simulated vernal equinox, prior to photoinduced increases in prolactin, thyrotropin-stimulating hormone-β and testicular growth. Analyses showed sustained elevated levels of daily FSHβ expression in the pituitary under conditions of the vernal, compared to autumnal equinox, short (&lt;12L) and long (&gt;12L) photoperiods. FSHβ expression increased in Quail held in non-stimulatory photoperiod, indicative of an endogenous programmed change in transcription. These data identify that FSHβ provides an endogenous program for the photoperiod-dependent external coincidence timing for seasonal transitions in reproduction. FSHβ may prime the pituitary for subsequent stimulation by triiodothyronine-mediated changes in tanycytes during the photoinducible phase. Overall, the data indicate a multi-cellular, multi-neural interval timing mechanism resides in the brain to control seasonal life-history transitions in birds

Evaluating and co-creating with student learning developers

This is a research article supported by a video. To read the article, please click on the pdf. To play the video, click on the link at the bottom of this page.The Learning Development Centre (LDC) in the School of Health Life Sciences at Glasgow Caledonian University serves over 6000 students, of whom over 70% have off-campus placement requirements. Provision of high-quality online support materials is therefore a priority. The LDC team has developed a suite of contextualised online resources to assist students in their development of academic and digital literacies. The LDC Team undertook a project to evaluate and enhance the resources and the LDC website. This involved the use of traditional evaluation tools in addition to the more innovative use of the 'Think Aloud Method' (Ericsson and Simon, 1984), to assess interface usability and online community content.A key to this project was the Teams partnership with students, as Student Learning Developers who played a pivotal role in the evaluation and co-creation of resources. This has helped to embed a culture of student collaboration within the LDC. In addition to showcasing the community and sharing students experiences of participating in the project, this submission will include discussion about effective ways of creating, evaluating and promoting online learning development resources, together with the LDC teams reflections on partnership working with students

Prolactin mediates long-term, seasonal rheostatic regulation of body mass in female mammals

A series of well-described anabolic and catabolic neuropeptides are known to provide short-term, homeostatic control of energy balance. The mechanisms that govern long-term, rheostatic control of regulated changes in energy balance are less well characterized. Using the robust and repeatable seasonal changes in body mass observed in Siberian hamsters, this report examined the role of prolactin in providing long-term rheostatic control of body mass and photoinduced changes in organ mass (ie, kidney, brown adipose tissue, uterine, and spleen). Endogenous circannual interval timing was observed after 4 months in a short photoperiod, indicated by a significant increase in body mass and prolactin mRNA expression in the pituitary gland. There was an inverse relationship between body mass and the expression of somatostatin (Sst) and cocaine- and amphetamine-regulated transcript (Cart). Pharmacological inhibition of prolactin release (via bromocriptine injection), reduced body mass of animals maintained in long photoperiods to winter–short photoperiod levels and was associated with a significant increase in hypothalamic Cart expression. Administration of ovine prolactin significantly increased body mass 24 hours after a single injection and the effect persisted after 3 consecutive daily injections. The data indicate that prolactin has pleiotropic effects on homeostatic sensors of energy balance (ie, Cart) and physiological effectors (ie, kidney, BAT). We propose that prolactin release from the pituitary gland acts as an output signal of the hypothalamic rheostat controller to regulate adaptive changes in body mass