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

Autophagic lysosome reformation dysfunction in glucocerebrosidase deficient cells: relevance to Parkinson disease.

Glucocerebrosidase (GBA1) gene mutations increase the risk of Parkinson disease (PD). While the cellular mechanisms associating GBA1 mutations and PD are unknown, loss of the glucocerebrosidase enzyme (GCase) activity, inhibition of autophagy and increased α-synuclein levels have been implicated. Here we show that autophagy lysosomal reformation (ALR) is compromised in cells lacking functional GCase. ALR is a cellular process controlled by mTOR which regenerates functional lysosomes from autolysosomes formed during macroautophagy. A decrease in phopho-S6K levels, a marker of mTOR activity, was observed in models of GCase deficiency, including primary mouse neurons and the PD patient derived fibroblasts with GBA1 mutations, suggesting that ALR is compromised. Importantly Rab7, a GTPase crucial for endosome-lysosome trafficking and ALR, accumulated in GCase deficient cells, supporting the notion that lysosomal recycling is impaired. Recombinant GCase treatment reversed ALR inhibition and lysosomal dysfunction. Moreover, ALR dysfunction was accompanied by impairment of macroautophagy and chaperone-mediated autophagy, increased levels of total and phosphorylated (S129) monomeric α-synuclein, evidence of amyloid oligomers and increased α-synuclein release. Concurrently, we found increased cholesterol and altered glucosylceramide homeostasis which could compromise ALR. We propose that GCase deficiency in PD inhibits lysosomal recycling. Consequently neurons are unable to maintain the pool of mature and functional lysosomes required for the autophagic clearance of α-synuclein, leading to the accumulation and spread of pathogenic α-synuclein species in the brain. Since GCase deficiency and lysosomal dysfunction occur with ageing and sporadic PD pathology, the decrease in lysosomal reformation may be a common feature in PD

Psychometric precision in phenotype definition is a useful step in molecular genetic investigation of psychiatric disorders.

Affective disorders are highly heritable, but few genetic risk variants have been consistently replicated in molecular genetic association studies. The common method of defining psychiatric phenotypes in molecular genetic research is either a summation of symptom scores or binary threshold score representing the risk of diagnosis. Psychometric latent variable methods can improve the precision of psychiatric phenotypes, especially when the data structure is not straightforward. Using data from the British 1946 birth cohort, we compared summary scores with psychometric modeling based on the General Health Questionnaire (GHQ-28) scale for affective symptoms in an association analysis of 27 candidate genes (249 single-nucleotide polymorphisms (SNPs)). The psychometric method utilized a bi-factor model that partitioned the phenotype variances into five orthogonal latent variable factors, in accordance with the multidimensional data structure of the GHQ-28 involving somatic, social, anxiety and depression domains. Results showed that, compared with the summation approach, the affective symptoms defined by the bi-factor psychometric model had a higher number of associated SNPs of larger effect sizes. These results suggest that psychometrically defined mental health phenotypes can reflect the dimensions of complex phenotypes better than summation scores, and therefore offer a useful approach in genetic association investigations.This work was supported by the Wellcome Trust [088869/Z/09/Z to M.R., P.B.J., D.G, and T. J. C,]; Medical Research Council [MC_UU_12019/1 and MC_UU_12019/3 to A.W, D.G., M.R]. Dr. Barnett is an employee of Cambridge Cognition, Ltd. This work forms part of the NIHR CLAHRC EoE that PBJ directs and the NIHR Cambridge Biomedical Research Centre.This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by NPG

Assessing the Nature of the Distribution of Localised States in Bulk GaAsBi.

A comprehensive assessment of the nature of the distribution of sub band-gap energy states in bulk GaAsBi is presented using power and temperature dependent photoluminescence spectroscopy. The observation of a characteristic red-blue-red shift in the peak luminescence energy indicates the presence of short-range alloy disorder in the material. A decrease in the carrier localisation energy demonstrates the strong excitation power dependence of localised state behaviour and is attributed to the filling of energy states furthest from the valence band edge. Analysis of the photoluminescence lineshape at low temperature presents strong evidence for a Gaussian distribution of localised states that extends from the valence band edge. Furthermore, a rate model is employed to understand the non-uniform thermal quenching of the photoluminescence and indicates the presence of two Gaussian-like distributions making up the density of localised states. These components are attributed to the presence of microscopic fluctuations in Bi content, due to short-range alloy disorder across the GaAsBi layer, and the formation of Bi related point defects, resulting from low temperature growth

Acidic environments trigger intracellular H+-sensing FAK proteins to re-balance sarcolemmal acid-base transporters and auto-regulate cardiomyocyte pH

AIMS: In cardiomyocytes, acute disturbances to intracellular pH (pHi) are promptly corrected by a system of finely-balanced sarcolemmal acid-base transporters. However, these fluxes become thermodynamically re-balanced in acidic environments, which inadvertently causes their set-point pHi to fall outside the physiological range. It is unclear whether an adaptive mechanism exists to correct this thermodynamic challenge and return pHi to normal. METHODS AND RESULTS: Following left-ventricle cryo-damage, a diffuse pattern of low extracellular pH (pHe) was detected by acid-sensing pHLIP. Despite this, pHi measured in the beating heart (13C NMR) was normal. Myocytes had adapted to their acidic environment by reducing Cl–/HCO3- exchange (CBE)-dependent acid-loading and increasing Na+/H+ exchange (NHE1)-dependent acid-extrusion, as measured by fluorescence (cSNARF1). The outcome of this adaptation on pHi is revealed as a cytoplasmic alkalinisation when cells are superfused at physiological pHe. Conversely, mice given oral bicarbonate to improve systemic buffering had reduced myocardial NHE1 expression, consistent with a needs-dependent expression of pHi-regulatory transporters. The response to sustained acidity could be replicated in vitro using neonatal ventricular myocytes (NRVMs) incubated at low pHe for 48 h. The adaptive increase in NHE1 and decrease in CBE activities was linked to Slc9a1 (NHE1) upregulation and Slc4a2 (AE2) downregulation. This response was triggered by intracellular H+ ions because it persisted in the absence of CO2/HCO3- and became ablated when acidic incubation media had low chloride concentration, a manoeuvre that reduces the extent of pHi decrease. Pharmacological inhibition of FAK-family non-receptor kinases, previously characterised as pH-sensors, ablated pHi autoregulation. In support of a pHi-sensing role, FAK protein Pyk2 (auto)phosphorylation was reduced within minutes of exposure to acidity, ahead of adaptive changes to pHi control. CONCLUSIONS: Cardiomyocytes fine-tune the expression of pHi-regulators so that pHi is at least 7.0. This autoregulatory feedback mechanism defines physiological pHi and protects it during pHe vulnerabilities. TRANSLATIONAL PERSPECTIVE: As a consequence of the inherent thermodynamic coupling between intra- and extracellular pH (pHi/pHe), sustained changes to perfusion, such as those in coronary disease or development, would have deleterious effects on the internal acid-base milieu of myocytes and hence cardiac function, unless offset by a corrective process. Using in-vivo and in-vitro models of acidification, we characterise this adaptive process functionally, and describe how it is engaged to auto-regulate pHi. This additional layer of homeostatic oversight enables the myocardium to operate within its optimal pHi-range, even at times when vascular perfusion is failing to maintain chemical constancy of the interstitial fluid

Preproglucagon Neurons in the Nucleus of the Solitary Tract are the Main Source of Brain GLP-1, Mediate Stress-Induced Hypophagia, and Limit Unusually Large Intakes of Food

Centrally administered glucagon-like peptide-1 (GLP-1) supresses food intake. Here we demonstrate that GLP-1-producing (PPG) neurons in the nucleus tractus solitarii (NTS) are the predominant source of endogenous GLP-1 within the brain. Selective ablation of NTS PPG neurons by viral expression of diphtheria toxin subunit A (DTA) substantially reduced active GLP-1 concentrations in brain and spinal cord. Contrary to expectations, this loss of central GLP-1 had no significant effect on ad libitum feeding of mice, affecting neither daily chow intake nor body weight or glucose tolerance. Only after bigger challenges to homeostasis were PPG neurons necessary for food intake control. PPG-ablated mice increased food intake following a prolonged fast and after a liquid diet preload. Consistent with our ablation data, acute inhibition of hM4Di-expressing PPG neurons did not affect ad libitum feeding, however, it increased post-fast refeeding intake and blocked stress-induced hypophagia. Additionally, chemogenetic PPG neuron activation through hM3Dq caused a strong acute anorectic effect. We conclude that PPG neurons are not involved in primary intake regulation, but form part of a secondary satiation/satiety circuit, activated by both psychogenic stress and large meals. Given their hypophagic capacity, PPG neurons might be an attractive drug target in obesity treatment

Forming next-generation antibody–nanoparticle conjugates through the oriented installation of non-engineered antibody fragments

The successful development of targeted nanotherapeutics is contingent upon the conjugation of therapeutic nanoparticles to target-specific ligands, with particular emphasis being placed on antibody-based ligands. Thus, new methods that enable the covalent and precise installation of targeting antibodies to nanoparticle surfaces are greatly desired, especially those which do not rely on costly and time-consuming antibody engineering techniques. Herein we present a novel method for the highly controlled and oriented covalent conjugation of non-engineered antibody F(ab) fragments to PLGA–PEG nanoparticles using disulfide-selective pyridazinedione linkers and strain-promoted alkyne–azide click chemistry. Exemplification of this method with trastuzumab and cetuximab showed significant improvements in both conjugation efficiency and antigen binding capability, when compared to commonly employed strategies for antibody–nanoparticle construction. This new approach paves the way for the development of antibody-targeted nanomedicines with improved paratope availability, reproducibility and uniformity to enhance both biological activity and ease of manufacture

Oriented attachment of VNAR proteins, via site-selective modification, on PLGA-PEG nanoparticles enhances nanoconjugate performance

Herein we report the construction of a nanoparticle-based drug delivery system which targets a key regulator in tumour angiogenesis. We exploit a Variable New Antigen Receptor (VNAR) domain, conjugated using site-specific chemistry, to direct poly lactic acid-co-glycolic acid-polyethylene glycol (PLGA-PEG) nanoparticles to delta like canonical Notch ligand 4 (DLL4). The importance of site-specific chemistry is demonstrated

Genes Suggest Ancestral Colour Polymorphisms Are Shared across Morphologically Cryptic Species in Arctic Bumblebees

email Suzanne orcd idCopyright: © 2015 Williams et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

On the Evolutionary Modification of Self-Incompatibility: Implications of Partial Clonality for Allelic Diversity and Genealogical Structure

Experimental investigations of homomorphic self-incompatibility (SI) have revealed an unanticipated level of complexity in its expression, permitting fine regulation over the course of a lifetime or a range of environmental conditions. Many flowering plants express some level of clonal reproduction, and phylogenetic analyses suggest that clonality evolves in a correlated fashion with SI in Solanum (Solanaceae). Here, we use a diffusion approximation to explore the effects on the evolutionary dynamics of SI of vegetative propagation with SI restricted to reproduction through seed. While clonality reduces the strength of frequency-dependent selection maintaining S-allele diversity, much of the great depth typical of S-allele genealogies is preserved. Our results suggest that clonality can play an important role in the evolution of SI systems, and may afford insight into unexplained features of allele genealogies in the Solanaceae