Interrogating surface intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates.

Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labelling sites. Here we describe a novel approach to improve extracellular labelling by functionalizing the SNAP-tag substrate benzyl guanine ("BG") with a charged sulfonate ("SBG").

Author Correction: Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics.

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label.

Conditional and Reversible Activation of Class A and B G Protein-Coupled Receptors Using Tethered Pharmacology.

Understanding the activation and internalization of G protein-coupled receptors (GPCRs) using conditional approaches is paramount to developing new therapeutic strategies. Here, we describe the design, synthesis, and testing of , a benzylguanine-linked peptide agonist of the glucagon-like peptide-1 receptor (GLP-1R), a class B GPCR required for maintenance of glucose levels in humans. covalently binds to SNAP-tagged GLP-1R-expressing cells, leading to prolonged cAMP generation, Ca rises, and intracellular retention of the receptor.

Potent Prearranged Positive Allosteric Modulators of the Glucagon-like Peptide-1 Receptor.

Drugs that allosterically modulate G protein-coupled receptor (GPCR) activity display higher specificity and may improve disease treatment. However, the rational design of compounds that target the allosteric site is difficult, as conformations required for receptor activation are poorly understood. Guided by photopharmacology, a set of prearranged positive allosteric modulators (PAMs) with restricted degrees of freedom was designed and tested against the glucagon-like peptide-1 receptor (GLP-1R), a GPCR involved in glucose homeostasis.

RAMP2 Influences Glucagon Receptor Pharmacology via Trafficking and Signaling.

Endogenous satiety hormones provide an attractive target for obesity drugs. Glucagon causes weight loss by reducing food intake and increasing energy expenditure. To further understand the cellular mechanisms by which glucagon and related ligands activate the glucagon receptor (GCGR), we investigated the interaction of the GCGR with receptor activity modifying protein (RAMP)2, a member of the family of receptor activity modifying proteins.

The preanalytical stability of glucagon as measured by liquid chromatography tandem mass spectrometry and two commercially available immunoassays.

Background One of the main challenges in the measurement of glucagon is the premise that it is unstable in human plasma. Traditionally, protease inhibitors have been used to prevent its degradation; however, their use is controversial. Here, we investigated the optimal method of sample collection for glucagon, with measurement by liquid chromatography tandem mass spectrometry (LC-MS/MS) and two commercially available immunoassays.

Allosteric Optical Control of a Class B G-Protein-Coupled Receptor.

Allosteric regulation promises to open up new therapeutic avenues by increasing drug specificity at G-protein-coupled receptors (GPCRs). However, drug discovery efforts are at present hampered by an inability to precisely control the allosteric site. Herein, we describe the design, synthesis, and testing of PhotoETP, a light-activated positive allosteric modulator of the glucagon-like peptide-1 receptor (GLP-1R), a class B GPCR involved in the maintenance of glucose homeostasis in humans.

Optical Control of Insulin Secretion Using an Incretin Switch.

Incretin mimetics are set to become a mainstay of type 2 diabetes treatment. By acting on the pancreas and brain, they potentiate insulin secretion and induce weight loss to preserve normoglycemia. Despite this, incretin therapy has been associated with off-target effects, including nausea and gastrointestinal disturbance.

The New Era of Drug Therapy for Obesity: The Evidence and the Expectations.

There is an urgent need for effective pharmacological therapies to help tackle the growing obesity epidemic and the healthcare crisis it poses. The past 3 years have seen approval of a number of novel anti-obesity drugs. The majority of these influence hypothalamic appetite pathways via dopaminergic or serotoninergic signalling.

Immunosuppression by mesenchymal stromal cells: from culture to clinic.

Extensive in vitro studies have shown that multipotent mesenchymal stromal cells (MSC) can exert profound immunosuppressive effects via modulation of both cellular and innate immune pathways. Their ability to be readily isolated from a number of tissues and expanded ex vivo makes them attractive candidates for systemic immunosuppressive therapy. In this article, we will review recent experimental data on the mechanisms by which MSC inhibit the alloproliferative response and the clinical relevance for their potential use in hematopoietic stem cell transplantation, solid organ transplantation, and treatment of autoimmune diseases.

Characterization of a mesenchymal-like stem cell population from osteophyte tissue.

Osteophytes are a distinct feature of osteoarthritis (OA). Their formation may be related to pluripotential cells in the periosteum responding to stimulus during OA. This study aimed to isolate stem cells from osteophyte tissues and to characterize their phenotype, proliferation, and differentiation potential, as well as their immunomodulatory properties.

Dynamic two-axis curvature measurement using multicore fiber Bragg gratings interrogated by arrayed waveguide gratings.

We describe the use of arrayed waveguide gratings (AWGs) in the interrogation of fiber Bragg gratings (FBGs) for dynamic strain measurement. The ratiometric AWG output was calibrated in a static deflection experiment over a +/-200 microepsilon range. Dynamic strain measurement was demonstrated with a FBG in a conventional single-mode fiber mounted on the surface of a vibrating cantilever and on a piezoelectric actuator, giving a resolution of 0.

Postnatal epithelial growth of the small intestine in the rat occurs by both crypt fission and crypt hyperplasia.

Studies of growth of the small intestine have largely concentrated on crypt hyperplasia rather than crypt fission. The aim of this study was to investigate quantitatively both crypt fission and crypt hyperplasia. DAxPVG/c rats were killed at 7, 11, 14, 17, 19, 21, 25, 55, and 72-73 days of life.