Journal: bioRxiv

Article Title: Gz Enhanced Signal Transduction assaY (G Z ESTY) for GPCR deorphanization

doi: 10.1101/2024.07.26.605282

Figure Lengend Snippet: ( A ) Concentration-response curves obtained for MOR stimulated with DAMGO, morphine, or β-endorphin. Data are shown as means ± SEM; N=3. ( B ) Concentration-response curves of endogenously expressed PAR1 activated with a selective agonist peptide TFLLR (left) or PAR2 activated with selective agonist peptide SLIGKV (right) in cells transfected only with the 3-in-one plasmid. Control cells were transfected with a 2-in-one plasmid not expressing GsGz chimera. Data are shown as means ± SEM; N=3. ( C ) Schematics of G z ESTY application to test ligand presence in mouse brain extract. Mouse brains were isolated after 10’’ of microwave exposure to preserve peptides and small molecules from degradation. Brain homogenates were sonicated, and debris and insoluble materials were separated by centrifugation. Supernatant was then applied to cells transfected with G z ESTY and resuspended in 96-well plates. cAMP levels were measured before and after brain extract application for a total of 25 minutes (created with BioRender). ( D ) Crude brain extract application induces activation of D2R, ADRA2A, MOR, and GABAB receptors. Representative traces (left) and quantification of 5 independent experiments (right) indicate the presence of endogenous ligands in the brain extract. Data are shown as means ± SEM; N=5. ( E ) Representative traces (left) and quantification (right) indicating the activation of GPR176 and GPR37 by crude brain extract application. Data are shown as means ± SEM; N=6.

Article Snippet: The following chemicals were purchased: clonidine (Tocris), dopamine (Tocris), GABA (Tocris), serotonin (Tocris), 1-oleoyl lysophosphatidic acid (Tocris), DAMGO (MedChemExpress), TFLLR (MedChemExpress), human PAR-2 (1-6, SLIGKV) (MedChemExpress), human galanin (1-30) (MedChemExpress), IBMX (MedChemExpress), SEW2871 (MedChemExpress), somatostatin-14 (Cpc Scientific), human neuropeptide Y (13-36) (Cpc Scientific), neuropeptide FF (Thermo Scientific Chemicals), MK-6892 (MedChemExpress), 2-arachidonoyl glycerol (Cayman Chemicals), N-Formyl-Met-Leu-Phe (R&D systems), SNC80 (Adipogen), isobutyric acid (TCI chemicals), salvinorin A (ChromaDex Inc.), morphine (Mallinckrodt Chemical Company), human β-endorphin (Sigma-Aldrich), teriparatide (MedChemExpress), NDP-α-MSH (Phoenix Pharmaceuticals), AB-MECA (MedChemExpress), quinpirole (Tocris), calcitonin gene-related peptide (CGRP) (AnaSpec).

Techniques: Concentration Assay, Transfection, Plasmid Preparation, Control, Expressing, Isolation, Sonication, Centrifugation, Activation Assay

Journal:

Article Title: Prenatal ?-Endorphin as an Early Predictor of Postpartum Depressive Symptoms in Euthymic Women

doi: 10.1016/j.jad.2009.12.009

Figure Lengend Snippet: β-Endorphin and Postpartum Depression in Women who are Euthymic (A) or Symptomatic (B) at 25 Weeks’ Gestational Age.

Article Snippet: Plasma levels of β-endorphin were determined by a direct solid phase two-site immunoradiometric assay (IRMA) using antibodies against synthetic human β-endorphin (Nichols Institute Diagnostics; San Juan Capistrano, California).

Techniques:

Journal: Nature Communications

Article Title: Femtosecond X-ray coherent diffraction of aligned amyloid fibrils on low background graphene

doi: 10.1038/s41467-018-04116-9

Figure Lengend Snippet: Preparation of TMV filaments and amyloid protofibrils on graphene. a , b Representative negative-stain TEM and AFM images of TMV, c – d bombesin filaments, and e , f β-endorphin filaments are shown. Negative-stain images ( a , c , e ) were acquired on fibrils placed on amorphous carbon films and AFM images ( b , d , f ) on graphene. Scalebars are 100 nm. b TMV fibrils align naturally on graphene over hundreds of nanometers. However, on the micrometer scale, aligned and randomly ordered fibrils are co-present. c Bombesin protofibrils associate laterally to form fibers, which randomly twist. A single preparation may consist of different polymorphs, e.g., twisted fibers and fibril rafts which are depicted here with arrows and squares, respectively. Bombesin fibers were mixed with TMV to compare their thickness. d The alignment of bombesin protofibrils on graphene is shown. Mature fibers are detected at larger magnifications. e β–endorphin protofibrils associate laterally to form twisted and striated fibers. f Aligned β–endorphin protofibrils were observed on graphene supports. To confirm that the features that are being imaged by the AFM are from the sample and not an artifact caused by the probe, the sample was rotated by 30 ° with respect to the scanning direction. Dashed circles represent the XFEL focus with FWHM = 150 nm

Article Snippet: The amyloid samples presented in the manuscript were purchased from BACHEM. β-endorphin (human) trifluoroacetate salt with the sequence: YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE is available under the catalog number H-2700.

Techniques: Staining

Journal: Nature Communications

Article Title: Femtosecond X-ray coherent diffraction of aligned amyloid fibrils on low background graphene

doi: 10.1038/s41467-018-04116-9

Figure Lengend Snippet: XFEL Diffraction patterns obtained from amyloid fibrils. Fibrils composed of bombesin and β-endorphin are shown on the left and right, respectively. a – b Single diffraction snapshots from aligned protofibrils, and background-subtracted merged patterns obtained from 40 diffraction snapshots each of c bombesin and d β-endorphin are shown. e , f Averaged intensity profiles as a function of reciprocal resolution over a band of width eight pixels (( e ) bombesin) and 22 pixels (( f ) β-endorphin) centered on the equator. Peaks in the equatorial profiles are marked. All peaks are summarized in Table

Article Snippet: The amyloid samples presented in the manuscript were purchased from BACHEM. β-endorphin (human) trifluoroacetate salt with the sequence: YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE is available under the catalog number H-2700.

Techniques:

Journal: Nature Communications

Article Title: Femtosecond X-ray coherent diffraction of aligned amyloid fibrils on low background graphene

doi: 10.1038/s41467-018-04116-9

Figure Lengend Snippet: Comparison of conventional X-ray patterns to merged XFEL patterns. Diffraction patterns from amyloid fibers composed of a Aβ(1–42) , b IAPP(1–37) , c Aβ(11–25) , and d Het-s(218-289) are shown. The equator and the most prominent layer lines are marked on the right side. The white and black arrow mark the meridional reflection at about 4.8 Å and the equatorial reflection at about ~10 Å characteristic for stacked β-sheets and present in ( a – d ), respectively. Note, that all amyloid fibrils are non-crystalline except for ( c ), which is crystalline. Merged XFEL diffraction patterns of bombesin ( e ) and β-endorphin fibrils ( f ) extending to 2.4 Å resolution are shown for qualitative comparison. a – c are reprinted from publication , Copyright (2010), with permission from Elsevier. d is reprinted from publication ( https://pubs.acs.org/doi/abs/10.1021%2Fbi5002807 ). Further permissions related to the material excerpted should be directed to the ACS

Article Snippet: The amyloid samples presented in the manuscript were purchased from BACHEM. β-endorphin (human) trifluoroacetate salt with the sequence: YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE is available under the catalog number H-2700.

Techniques: