New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Interleukin-4 clicked surfaces drive M2 macrophage polarization

Driving macrophage (Mφ) polarization into the M2 phenotype provides exciting potential against various inflammatory diseases. Interleukin-4 (IL-4) leverages polarization into the M2-Mφ phenotype but its systemic use is constrained by dose-limiting toxicity. Consequently, we developed IL-4 decorated surfaces aiming for sustained and localized activity of the potent cytokine. IL-4 muteins were generated through genetic code expansion, replacing Lys 42 by unnatural amino acids (uAA). All muteins had wt-IL-4 comparable cellular performances and binding affinities to the IL4Rα. Copper catalyzed (CuAAC) and copper free strain promoted (SPAAC) [1+3]-dipolar azide alkyne cycloadditions were used to site-selectively anchor IL-4 at position 42 to agarose surfaces. The IL4-decorated surfaces provided sustained IL-4 activity as demonstrated by TF-1 cell proliferation and by M2 but not M1 polarization of M-CSF generated human Mφ. The approach demonstrated for IL-4 provides a blueprint for the engineering of cytokine-activated surfaces profiled for sustained and spatially controlled activity

Proton detection and breathing regulation by the retrotrapezoid nucleus

We discuss recent evidence which suggests that the principal central respiratory chemoreceptors are located within the retrotrapezoid nucleus (RTN) and that RTN neurons are directly sensitive to [H+]. RTN neurons are glutamatergic. In vitro, their activation by [H+] requires expression of a proton-activated GPCR (GPR4) and a proton-modulated potassium channel (TASK-2). These proteins are undetectable in astrocytes and the rest of the lower brainstem respiratory network. The pH response of RTN neurons is modulated by surrounding astrocytes but genetic deletion of RTN neurons or deletion of both GPR4 and TASK-2 virtually eliminates the central respiratory chemoreflex. Thus, although this reflex is regulated by innumerable brain pathways, it seems to operate predominantly by modulating the discharge rate of RTN neurons, and the activation of RTN neurons by hypercapnia may ultimately derive from their intrinsic pH sensitivity. RTN neurons increase lung ventilation by stimulating multiple aspects of breathing simultaneously. They stimulate breathing about equally during quiet wake and non-REM sleep and to a lesser degree during REM sleep. The activity of RTN neurons is regulated by inhibitory feedback and by excitatory inputs, notably from the carotid bodies. The latter input operates during normo- or hypercapnia but fails to activate RTN neurons under hypocapnic conditions. RTN inhibition likely limits the degree of hyperventilation produced by hypobaric hypoxia. RTN neurons are also activated by inputs from serotonergic neurons and hypothalamic neurons. The absence of RTN neurons probably underlies the sleep apnea and lack of chemoreflex that characterize congenital central hypoventilation syndrome

Peptide Activation of Epithelial Adgrf5 (Gpr116) Regulates Pulmonary Alveolar Homeostasis

Pulmonary alveolar homeostasis is dependent upon balanced airway and tissue surfactant pools. Quantitative and qualitative alterations in alveolar surfactant pools are associated with inflammation and tissue destruction in severe lung diseases including infant respiratory distress syndrome, acute lung injury and pulmonary alveolar proteinosis. Identification of a physiologically-dominant molecular pathway within alveolar epithelial cells that senses and regulates endogenous alveolar surfactant pools, coupled with the ability to pharmacologically modulate it both positively and negatively, would be a major therapeutic advance for patients with lung diseases associated with pulmonary surfactant disorders. We and others have previously shown that Gpr116 is a critical regulator of surfactant homeostasis in mice. Here we extend this work to show that human and mouse Gpr116 proteins are highly conserved at the amino acid level, are expressed on the plasma membrane of alveolar type II cells and functionally couple to intracellular G proteins when activated. Further, we have identified a synthetic peptide, GAP16, that is capable of activating mouse and human Gpr116 in vitro, resulting in increased Gq/11-dependent inositol phosphate conversion and calcium mobilization, cortical F-actin stabilization, and increased impedance of cell monolayers. Administration of GAP16 suppressed surfactant secretion from primary type II cells in vitro and nebulization of GAP16 to wild type mice was sufficient to suppress surfactant secretion from alveolar type II cells in vivo. These data provide proof-of-concept that Gpr116 is a plausible therapeutic target to modulate endogenous alveolar surfactant pools in humans to pulmonary diseases associated with surfactant dysfunction

The proton-activated receptor GPR4 contributes to central respiratory chemosensitivity

The ability to sense CO2/H+ is critical for normal breathing. A specific group of neurons within the medullary retrotrapezoid nucleus (RTN), identified by Phox2b-expression, are intrinsically sensitive to CO2/H+ and are preeminent central respiratory chemosensory neurons1,2. These excitatory neurons stimulate ventilation to rapidly regulate CO2 excretion and acid-base balance3 and they are selectively ablated in a mouse genetic model of human congenital central hypoventilation syndrome (CCHS) that recapitulates the blunted CO2-induced breathing and increased central apneas of CCHS4-6. However, the molecular mechanisms that mediate pH sensing in these neurons remain incompletely understood. Here, we demonstrate that GPR4, a proton-activated G protein-coupled receptor, represents a molecular substrate for CO2/H+-dependent regulation of RTN neuronal excitability and central respiratory chemosensitivity. Ventilatory stimulation by raised CO2, but not lowered O2, was strongly reduced in GPR4 knockout mice, which were also more prone to apneic events during quiet hyperoxic breathing. Mice lacking GPR4 showed a striking reduction in CO2 activation of RTN neurons in vivo, as determined by cFos expression. Likewise, RTN neuronal excitability and pH sensitivity in vitro were reduced by GPR4 deletion or receptor blockade, and modulated by intracellular application of GTP analogs. Finally, re-expression of GPR4 into RTN neurons of GPR4-/- mice rescued CO2-stimulated cFos expression and ventilation, and returned apneic frequencies to wild type levels. Taken together, this work identifies a new role for GPR4, defines a novel molecular component of central respiratory chemosensitivity, and suggests new therapeutic options to regulate breathing

Receptors for protons or lipid messengers or both?

The subfamily of G protein-coupled receptors comprising GPR4, OGR1, TDAG8, and G2A was originally characterized as a group of proteins mediating biological responses to the lipid messengers sphingosylphosphorylcholine (SPC), lysophosphatidylcholine (LPC), and psychosine. We challenged this view by reporting that OGR1 and GPR4 sense acidic pH and that this process is not affected by concentrations of SPC or LPC previously reported as agonistic. The original publications describing GPR4, OGR1, and G2A as receptors for LPC or SPC have now been retracted, and the first studies exploring receptors of this family as pH sensors in physiology have appeared. Here we review the status of this field and we confirm that GPR4, OGR1, and TDAG8 should be considered as proton-sensing receptors. Negative regulation of these receptors by high micromolar concentrations of lipids appears not specific in our experiments

The G protein-coupled pH-sensing receptor OGR1 is a regulator of intestinal inflammation

Background: A novel family of proton sensing G-protein coupled receptors (GPCRs), including ovarian cancer G-protein coupled receptor 1 (OGR1, GPR68), GPR4, and T-cell death associated gene 8 (TDAG8, GPR65), was previously identified to play an important role in physiological pH homeostasis. Luminal and tissue pH values in patients with active inflammatory bowel disease (IBD) are known to be significantly lower than values in the normal subject. We investigated whether OGR1 may play a role for the pathophysiology of IBD. Methods: OGR1 expression in colonic tissues was investigated in controls and IBD patients. Expression of OGR1 upon cell activation was studied in the Mono Mac 6 (MM6) cell line and primary human and murine monocytes by real-time PCR. Ogr1 knockout mice were crossbred with Il-10 deficient mice, compared to Il-10 -/- from the same litters and studied over 200 days. Microarray profiling (Metacore GeneGo and ranked fold change analysis) was performed using Ogr1-/- and Ogr1+/+ (WT) residential peritoneal macrophages. Results: IBD patients expressed higher levels of OGR1 in the mucosa as compared to controls. Treatment of MM6 cells with TNF, but not other cytokines (IFN-γ, IL-1β, IL-6, TGF-β) tested, led to significant up-regulation of OGR1 mRNA expression. Induction of OGR1 expression by TNF was dose-dependent. Macrophage differentiation of MM6 cells with PMA also led to a significant increase in OGR1 expression. Dose-dependence of TNF and PMA mediated induction of OGR1 expression was confirmed in primary human monocytes. TNF-mediated induction of OGR1 mRNA expression was reversed by simultaneous treatment of cells with the NF-κB inhibitors MG132, AICAR, BAY-11-7082, CAY10512, and SC-514. Kaplan-Meier survival analysis showed a significantly delayed onset and progression of rectal prolapse in female Ogr1-/- // Il-10-/- mice (p=0.005). Female Ogr1 -/- // Il-10 -/- mice had significantly less rectal prolapses (21%, n = 19 versus 75%, n = 8; p = 0.025; Odd ratio = 0.089). Up-regulation of gene expression, mediated by OGR1, in response to extracellular acidification in mouse macrophages was enriched for inflammation and immune response, actin cytoskeleton, and cell adhesion gene pathways. Conclusion: OGR1 expression is induced in cells of human macrophage lineage and primary human monocytes by TNF and PMA. NF-κB inhibition reverses the induction of OGR1 mRNA expression by TNF. OGR1 deficiency protects from spontaneous inflammation in the Il-10 KO model. Our data indicate an important pathophysiological role for the pH sensing G-protein coupled receptor OGR1 during the pathogenesis of mucosal inflammation

Exploring Glucocorticoid Receptor Agonists Mechanism of Action Through Mass Cytometry and Radial Visualizations

Recent advances in combining flow cytometry and mass spectrometry have led to the development of mass cytometry, allowing for the interrogation of complex cell populations on an unprecedented scale. The volumes and high dimensionality of mass cytometry data pose significant challenges in terms of analysis and visualization. We implement a method called Radviz, where multidimensional single cell data can be visualized as a projection that maintains the original dimensions and data complexity whilst facilitating analysis and visualization. This enables identification of changes in populations, focusing the analysis on the most relevant aspect of large multidimensional datasets. To highlight the potential of Radviz, we profiled peripheral mononuclear blood cells (PBMCs) from three healthy donors and showed donor-specific differences in the number and composition of cell populations. In a second study, we explored the anti-inflammatory effects of two glucocorticoid receptor (GR) ligands (cpd6 and cpd11) compared to dexamethasone (Dex) on human primary macrophages. Standard analysis at the population level showed that cpd6 and cpd11 have an overall anti-inflammatory profile similar to that of Dex. CyTOF profiling and Radviz-driven analysis at the single cell level confirmed this observation, and identified a concentration-dependent effect of cpd6 that was not detected at the population level. Altogether, Radviz combines the strengths of a projection method, reducing the dimensionality of datasets, with that of a scatter plot, where the identity of each point can be inferred from the distance to the axis. This enables the visual exploration, analysis, and interpretation of complex, high dimensional data