Distribution and characterisation of Glucagon-like peptide-1 receptor expressing cells in the mouse brain.

© 2015 The Authors.Objective: Although Glucagon-like peptide 1 is a key regulator of energy metabolism and food intake, the precise location of GLP-1 receptors and the physiological relevance of certain populations is debatable. This study investigated the novel GLP-1R-Cre mouse as a functional tool to address this question. Methods: Mice expressing Cre-recombinase under the Glp1r promoter were crossed with either a ROSA26 eYFP or tdRFP reporter strain to identify GLP-1R expressing cells. Patch-clamp recordings were performed on tdRFP-positive neurons in acute coronal brain slices from adult mice and selective targeting of GLP-1R cells in vivo was achieved using viral gene delivery. Results: Large numbers of eYFP or tdRFP immunoreactive cells were found in the circumventricular organs, amygdala, hypothalamic nuclei and the ventrolateral medulla. Smaller numbers were observed in the nucleus of the solitary tract and the thalamic paraventricular nucleus. However, tdRFP positive neurons were also found in areas without preproglucagon-neuronal projections like hippocampus and cortex. GLP-1R cells were not immunoreactive for GFAP or parvalbumin although some were catecholaminergic. GLP-1R expression was confirmed in whole-cell recordings from BNST, hippocampus and PVN, where 100 nM GLP-1 elicited a reversible inward current or depolarisation. Additionally, a unilateral stereotaxic injection of a cre-dependent AAV into the PVN demonstrated that tdRFP-positive cells express cre-recombinase facilitating virally-mediated eYFP expression. Conclusions: This study is a comprehensive description and phenotypic analysis of GLP-1R expression in the mouse CNS. We demonstrate the power of combining the GLP-1R-CRE mouse with a virus to generate a selective molecular handle enabling future in vivo investigation as to their physiological importance

Limited impact on glucose homeostasis of leptin receptor deletion from insulin- or proglucagon-expressing cells

Aims/hypothesis The adipose tissue-derived hormone leptin plays an important role in the maintenance of body weight and glucose homeostasis. Leptin mediates its effects by interaction with leptin receptors (LepRb), which are highly expressed in the hypothalamus and other brain centres, and at lower levels in the periphery. Previous studies have used relatively promiscuous or inefficient Cre deleter strains, respectively, to explore the roles of LepR in pancreatic β and α cells. Here, we use two newly-developed Cre lines to explore the role of leptin signalling in insulin and proglucagon-expressing cells. Methods Leptin receptor expression was measured in isolated mouse islets and highly-purified islet cells by RNASeq and quantitative RT-PCR. Mice lacking leptin signalling in pancreatic β, or in α and other proglucagon-expressing cells, were generated using Ins1Cre- or iGluCre-mediated recombination respectively of flox'd leptin receptor alleles. In vivo glucose homeostasis, changes in body weight, pancreatic histology and hormone secretion from isolated islets were assessed using standard techniques. Results Leptin receptor mRNA levels were at or below the level of detection in wild-type adult mouse isolated islets and purified cells, and leptin signalling to Stat3 phosphorylation was undetectable. Whereas male mice further deleted for leptin receptors in β cells exhibited no abnormalities in glucose tolerance up to 16 weeks of age, females transiently displayed improved glucose tolerance at 8 weeks (11.2 ± 3.2% decrease in area under curve; p < 0.05), and improved (39.0 ± 13.0%, P < 0.05) glucose-stimulated insulin secretion in vitro. No differences were seen between genotypes in body weight, fasting glucose or β/α cell ratio. Deletion of LepR from α-cells, a minority of β cells, and a subset of proglucagon-expressing cells in the brain, exerted no effects on body weight, glucose or insulin tolerance, nor on pancreatic hormone secretion assessed in vivo and in vitro. Conclusions/interpretation The use here of a highly selective Cre recombinase indicates that leptin signalling plays a relatively minor, age- and sex-dependent role in the control of β cell function in the mouse. No in vivo role for leptin receptors on α cells, nor in other proglucagon-expressing cells, was detected in this study

UBE2QL1 is Disrupted by a Constitutional Translocation Associated with Renal Tumor Predisposition and is a Novel Candidate Renal Tumor Suppressor Gene

Investigation of rare familial forms of renal cell carcinoma (RCC) has led to the identification of genes such as VHL and MET that are also implicated in the pathogenesis of sporadic RCC. In order to identify a novel candidate renal tumor suppressor gene, we characterized the breakpoints of a constitutional balanced translocation, t(5;19)(p15.3;q12), associated with familial RCC and found that a previously uncharacterized gene UBE2QL1 was disrupted by the chromosome 5 breakpoint. UBE2QL1 mRNA expression was downregulated in 78.6% of sporadic RCC and, although no intragenic mutations were detected, gene deletions and promoter region hypermethylation were detected in 17.3% and 20.3%, respectively, of sporadic RCC. Reexpression of UBE2QL1 in a deficient RCC cell line suppressed anchorage-independent growth. UBE2QL1 shows homology to the E2 class of ubiquitin conjugating enzymes and we found that (1) UBE2QL1 possesses an active-site cysteine (C88) that is monoubiquitinated in vivo, and (2) UBE2QL1 interacts with FBXW7 (an F box protein providing substrate recognition to the SCF E3 ubiquitin ligase) and facilitates the degradation of the known FBXW7 targets, CCNE1 and mTOR. These findings suggest UBE2QL1 as a novel candidate renal tumor suppressor gen

Preproglucagon neurons in the hindbrain have IL-6 Receptor α (IL-6Rα) and show Ca 2+ influx in response to IL-6

Neuronal circuits in the hypothalamus and hindbrain are of importance for control of food intake, energy expenditure, and fat mass. We have recently shown that treatment with exendin-4 (Ex-4), an analog of the proglucagon-derived molecule glucagon-like peptide 1 (GLP-1), markedly increases mRNA expression of the cytokine interleukin-6 (IL-6) in the hypothalamus and hindbrain and that this increase partly mediates the suppression of food intake and body weight by Ex-4. Endogenous GLP-1 in the central nervous system (CNS) is produced by preproglucagon (PPG) neurons of the nucleus of the solitary tract (NTS) in the hindbrain. These neurons project to various parts of the brain, including the hypothalamus. Outside the brain, IL-6 stimulates GLP-1 secretion from the gut and pancreas. In this study, we aim to investigate whether IL-6 can affect GLP-1-producing PPG neurons in the nucleus of the solitary tract (NTS) in mouse hindbrain via the ligand binding part of the IL-6 receptor, IL-6 receptor-α (IL-6Rα). Using immunohistochemistry, we found that IL-6Rα was localized on PPG neurons of the NTS. Recordings of these neurons in GCaMP3/GLP-1 reporter mice showed that IL-6 enhances cytosolic Ca2+ concentration in neurons capable of expressing PPG. We also show that the Ca2+ increase originates from the extracellular space. Furthermore, we found that IL-6Rα was localized on cells in the caudal hindbrain expressing immunoreactive NeuN (a neuronal marker) or CNP:ase (an oligodendrocyte marker). In summary, IL-6Rα is present on PPG neurons in the NTS, and IL-6 can stimulate these cells by increasing influx of Ca2+ to the cytosol from the extracellular space

Operation Disaster Defender: A Multi-Disciplinary Approach to Preserving Access to Justice and Client Property through Disaster Preparedness

For centuries, the legal profession has prided itself on managing the impacts of crises and disasters for our clients. However, as disasters seem to be more commonplace than before, is the legal profession prepared to manage a crisis of its own? With the livelihood of nearly 79,0002 possibly at stake in the United States, shouldn\u27t preparing for a disaster be one of our top priorities as a profession? Additionally, the professional responsibility rules and civil liability will likely not be completely suspended during a disaster. This means that individuals could possibly face discipline or civil liability for failing to prepare for a disaster. Though this culture of preparedness is growing; what about our duty as leaders to preparing our communities? Would it be surprising to learn that there has been a gap in collaboration between legal and emergency management profession until it\u27s too late? Though some work has been done to help bridge this gap, more can still be done. The Emergency Management profession has developed and routinely updates Emergency Management program accreditation and certification programs for itself that are used in various industries both domestically and internationally. Incorporating these established programs within the legal community will allow the legal profession to have a solid foundation in mitigating professional responsibility and civil liability pitfalls following a disaster. Additionally, the tertiary effects of this program are likely to: (1) provide a ready and capable legal community who can easily understand and assist emergency managers and civic leaders with some of their most misunderstood legal problems before, during, and after a disaster; (2) safeguard access to justice after a disaster strikes; and (3) preserve client and third-party tangible personal property

It\u27s A Trap!: Responsible Enforcement of Texas Disaster Evacuation Orders

We see emergencies within our lives and communities every day without much fanfare. However, when emergencies impact entire neighborhoods, communities, or regions, they become disasters. Depending on its severity, residents can be forced to flee in search of safety. Texas has had nearly five times the annual average of federal disaster declarations than that of any other state in the union. To manage emergencies and disasters, Texas law affords local governments numerous powers, including the authority to order evacuations. While many states have a single mechanism to enforce evacuation orders, Texas has both a civil (recovery of rescue expenses) and criminal (misdemeanor offense) enforcement mechanism. While the criminal mechanism has a misdemeanor offense tied to it, the civil mechanism allows jurisdictions to recover the cost of a rescue from people who ignored an evacuation order. This Article examines Texas\u27s dual enforcement approach and the need for a statutory exception. This Article does not examine (1) whether mandatory evacuations constitute a Fifth Amendment taking or (2) whether a moral duty to rescue exists during a disaster

Mechanisms explaining transitions between tonic and phasic firing in neuronal populations as predicted by a low dimensional firing rate model

Several firing patterns experimentally observed in neural populations have been successfully correlated to animal behavior. Population bursting, hereby regarded as a period of high firing rate followed by a period of quiescence, is typically observed in groups of neurons during behavior. Biophysical membrane-potential models of single cell bursting involve at least three equations. Extending such models to study the collective behavior of neural populations involves thousands of equations and can be very expensive computationally. For this reason, low dimensional population models that capture biophysical aspects of networks are needed. \noindent The present paper uses a firing-rate model to study mechanisms that trigger and stop transitions between tonic and phasic population firing. These mechanisms are captured through a two-dimensional system, which can potentially be extended to include interactions between different areas of the nervous system with a small number of equations. The typical behavior of midbrain dopaminergic neurons in the rodent is used as an example to illustrate and interpret our results. \noindent The model presented here can be used as a building block to study interactions between networks of neurons. This theoretical approach may help contextualize and understand the factors involved in regulating burst firing in populations and how it may modulate distinct aspects of behavior.Comment: 25 pages (including references and appendices); 12 figures uploaded as separate file

Ten steps or climbing a mountain: A study of Australian health professionals' perceptions of implementing the baby friendly health initiative to protect, promote and support breastfeeding

Background: The Baby Friendly Hospital (Health) Initiative (BFHI) is a global initiative aimed at protecting, promoting and supporting breastfeeding and is based on the ten steps to successful breastfeeding. Worldwide, over 20,000 health facilities have attained BFHI accreditation but only 77 Australian hospitals (approximately 23%) have received accreditation. Few studies have investigated the factors that facilitate or hinder implementation of BFHI but it is acknowledged this is a major undertaking requiring strategic planning and change management throughout an institution. This paper examines the perceptions of BFHI held by midwives and nurses working in one Area Health Service in NSW, Australia. Methods: The study used an interpretive, qualitative approach. A total of 132 health professionals, working across four maternity units, two neonatal intensive care units and related community services, participated in 10 focus groups. Data were analysed using thematic analysis. Results: Three main themes were identified: ‘Belief and Commitment’; ‘Interpreting BFHI’ and ‘Climbing a Mountain’. Participants considered the BFHI implementation a high priority; an essential set of practices that would have positive benefits for babies and mothers both locally and globally as well as for health professionals. It was considered achievable but would take commitment and hard work to overcome the numerous challenges including a number of organisational constraints. There were, however, differing interpretations of what was required to attain BFHI accreditation with the potential that misinterpretation could hinder implementation. A model described by Greenhalgh and colleagues on adoption of innovation is drawn on to interpret the findings. Conclusion: Despite strong support for BFHI, the principles of this global strategy are interpreted differently by health professionals and further education and accurate information is required. It may be that the current processes used to disseminate and implement BFHI need to be reviewed. The findings suggest that there is a contradiction between the broad philosophical stance and best practice approach of this global strategy and the tendency for health professionals to focus on the ten steps as a set of tasks or a checklist to be accomplished. The perceived procedural approach to implementation may be contributing to lower rates of breastfeeding continuation

An unusual flavin-dependent halogenase from the metagenome of the marine sponge Theonella swinhoei WA

The authors thank EU BlueGenics (Seventh Framework Programme, Collaborative project “BlueGenics”, Grant no. 311848 RJMG and JP), the SNF (Grant no.205321_165695 to JP), the Helmut Horten Foundation (JP), and ERAIB (Grant no. 031A338A KHVP and RJMG) for funding.Uncultured bacteria from sponges have been demonstrated to be responsible for the generation of many potent, bioactive natural products including halogenated metabolites.1 The identification of gene clusters from the metagenomes of such bacterial communities enables the discovery of enzymes that mediate new and useful chemistries and allows insight to be gained into the biogenesis of potentially pharmacologically important natural products. Here we report a new pathway to the keramamides (krm); the first functional evidence for the existence of a distinct producer in the Theonella swinhoei WA chemotype is revealed, and a key enzyme on the pathway, a unique flavin dependent halogenase with a broad substrate specificity, and with potential as a useful new biocatalytic tool is described.PostprintPeer reviewe

Massively Parallel Sequencing Reveals the Complex Structure of an Irradiated Human Chromosome on a Mouse Background in the Tc1 Model of Down Syndrome

Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype – phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1) presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2) overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439