Light microscopy combined with well-established protocols of two-dimensional cell culture facilitates high-throughput quantitative imaging to study biological phenomena. Accurate segmentation of individual cells in images enables exploration of complex biological questions, but can require sophisticated imaging processing pipelines in cases of low contrast and high object density. Deep learning-based methods are considered state-of-the-art for image segmentation but typically require vast amounts of annotated data, for which there is no suitable resource available in the field of label-free cellular imaging. Here, we present LIVECell, a large, high-quality, manually annotated and expert-validated dataset of phase-contrast images, consisting of over 1.6 million cells from a diverse set of cell morphologies and culture densities. To further demonstrate its use, we train convolutional neural network-based models using LIVECell and evaluate model segmentation accuracy with a proposed a suite of benchmarks.
A series of saligenin beta(2) adrenoceptor agonist antedrugs having high clearance were prepared by reacting a protected saligenin oxazolidinone with protected hydroxyethoxyalkoxyalkyl bromides, followed by removal of the hydroxy-protecting group, alkylation, and final deprotection. The compounds were screened for beta(2), beta(1), and beta(3) agonist activity in CHO cells. The onset and duration of action in vitro of selected compounds were assessed on isolated superfused guinea pig trachea. Compound 13f had high potency, selectivity, fast onset, and long duration of action in vitro and was found to have long duration in vivo, low oral bioavailability in the rat, and to be rapidly metabolized. Crystalline salts of 13f (vilanterol) were identified that had suitable properties for inhaled administration. A proposed binding mode for 13f to the beta(2)-receptor is presented.
A series of saligenin alkoxyalkylphenylsulfonamide beta(2) adrenoceptor agonists were prepared by reacting a protected saligenin oxazolidinone with alkynyloxyalkyl bromides, followed by Sonogashira reaction, hydrogenation, and deprotection. The meta-substituted primary sulfonamide was more potent than the para- and the ortho-analogues. Primary sulfonamides were more potent than the secondary and tertiary analogues. The onset and duration of action in vitro of selected compounds was assessed on isolated superfused guinea pig trachea. Sulfonamide 29b had the best profile of potency, selectivity, onset, and duration of action on both guinea pig trachea and human bronchus. Furthermore, 29b was found to have low oral bioavailability in rat and dog and also to have long duration of action in an in vivo model of bronchodilation. Crystalline salts of 29b were identified that had suitable properties for inhaled administration. A proposed binding mode for 29b to the beta(2)-receptor is presented.
GBRs (GABA(B) receptors; where GABA stands for gamma-aminobutyric acid) are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. In vitro assays have previously demonstrated that these receptors are heterodimers assembled from two homologous subunits, GBR1 and GBR2, neither of which is capable of producing functional GBR on their own. We have used co-immunoprecipitation in combination with bioluminescence and fluorescence resonance energy transfer approaches in living cells to assess directly the interaction between GBR subunits and determine their subcellular localization. The results show that, in addition to forming heterodimers, GBR1 and GBR2 can associate as stable homodimers. Confocal microscopy indicates that, while GBR1/GBR1 homodimers are retained in the endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartment, both GBR2/GBR2 homodimers and GBR1/GBR2 heterodimers are present at the plasma membrane. Although these observations shed new light on the assembly of GBR complexes, they raise questions about the potential functional roles of GBR1 and GBR2 homodimers.
Fusion proteins were generated by attachment of green fluorescent protein (GFP) to the C-terminal tail of either the wild-type human beta(2)-adrenoceptor or a form with enhanced constitutive activity. Sustained treatment of HEK293 cells stably expressing the constitutively active mutant (CAM) beta(2)-adrenoceptor-GFP with the inverse agonist betaxolol resulted in a marked up-regulation of the fusion protein that could be monitored by both fluorescence and immunoblotting of membrane fractions. This was not observed for the wild-type beta(2)-adrenoceptor-GFP. Addition of the agonist isoprenaline to CAM beta(2)-adrenoceptor-GFP expressing cells previously treated with betaxolol resulted in rapid internalization of the receptor into punctate intracellular vesicles in a manner similar to wild-type beta(2)-adrenoceptor-GFP. A range of "beta-blockers" replicated the up-regulation of the CAM beta(2)-adrenoceptor-GFP, although pharmacological specificity was maintained, as it was not produced by alpha(1)- and alpha(2)-adrenoceptor-selective antagonists/inverse agonists. Parallel intact cell binding studies with [(3)H]dihydroalprenolol confirmed up-regulation of the CAM beta(2)-adrenoceptor-GFP by betaxolol but failed to predict the optically monitored up-regulation produced by high concentrations of alprenolol. The cellular distribution of the up-regulated CAM beta(2)-adrenoceptor-GFP was not identical after sustained treatment of the cells with different beta-blockers. Inverse agonists, able to reduce basal intracellular cAMP levels, such as betaxolol and ICI118551, resulted in both increased plasma membrane receptor and increased diffuse intracellular staining. In contrast, treatment with labetolol and alprenolol resulted in a significant fraction of the intracellular receptor displaying a punctate distribution pattern. These ligands displayed substantial agonism to stimulate intracellular cAMP levels via the CAM beta(2)-adrenoceptor-GFP.
Constitutively active forms of the hamster alpha(1b)-adrenoceptor can be produced from the point mutations Asp(142)Ala or Ala(293)Glu or exchange of a small segment of the third intracellular loop with the equivalent region of the beta(2)-adrenoceptor. Green fluorescent protein (GFP)-tagged forms of each of these mutants and of the wild type alpha(1b)-adrenoceptor were expressed stably in HEK293 cells. The wild type alpha(1b)-adrenoceptor-GFP was expressed both at the plasma membrane and with a distinctly perinuclear punctate pattern. Sustained treatment with a range of antagonist/inverse agonist ligands failed to modulate the cellular distribution or levels of expression of this construct. The form of the alpha(1b)-adrenoceptor containing the beta(2)-adrenoceptor sequence substitution was predominantly located in punctate intracellular vesicles and sustained challenge with the same series of antagonists/inverse agonists produced a 5-fold up-regulation of protein levels with elevation of both plasma membrane and intracellular receptor. Quantification of these effects could be produced by spectrofluorometric analysis of cells grown in a 96-well microtiter plate. In contrast, both the Asp(142)Ala and Ala(293)Glu forms of the alpha(1b)-adrenoceptor-GFP were located predominantly at the plasma membrane. Levels of these two point mutants were not increased by any of the antagonist/inverse agonist ligands tested, although the sequence substitution mutation encompasses codon 293. Resolution of constitutive activity and ligand-induced up-regulation was further exemplified by a mutant lacking eight serine residues in the C-terminal tail that displayed little constitutive activity but was up-regulated by sustained ligand challenge. These results demonstrate the nonequivalence of mutations in their regulation by antagonist/inverse agonist ligands.
1 The b 2 -agonist salmeterol is a potent relaxant of airway smooth muscle with a long duration of action. Previous studies of cyclic AMP accumulation, however, have indicated that salmeterol is a low ecacy b 2 -agonist when compared to isoprenaline. Here we have compared the properties of salmeterol and isoprenaline as stimulants of gene transcription in CHO-K1 cells transfected with the human b 2 -adrenoceptor to dierent levels (50 and 310 fmol mg protein 71 ). 2 Gene transcription was monitored using a secreted placental alkaline phosphate (SPAP) reporter gene under the transcriptional control of six cyclic AMP response element (CRE) sequences. 3 In the lower expressing cells (CHO-b 2 /6), salmeterol produced a maximal cyclic AMP response that was only 22% that of that obtained with isoprenaline. In contrast in the higher expressing cells (CHO-b 2 / 4), the two maxima were of similar magnitude. 4 Salmeterol was a more potent stimulant of gene transcription, producing the same maximal response as isoprenaline in both cell lines. Furthermore, in the CHO-b 2 /4 cells, Salmeterol was 50 fold more potent as a stimulant of SPAP secretion than of cyclic AMP accumulation. In contrast, isoprenaline was 24 fold less sensitive as a stimulant of SPAP secretion than of cyclic AMP accumulation. In the presence of serum (10%), the eects of both salmeterol and isoprenaline on gene transcription were augmented. 5 These data suggest that the low ecacy and/or long duration of action of salmeterol, favours a potent stimulation of gene transcription when compared to more ecacious but shorter-lived agonists such as isoprenaline.
1 The wild-type b 2 -adrenoceptor and a constitutively active mutant of this receptor were Cterminally tagged with luciferase from the sea pansy Renilla reniformis. 2 C-terminal addition of Renilla luciferase did not substantially alter the levels of expression of either form of the receptor, the elevated constitutive activity of the mutant b 2 -adrenoceptor nor the capacity of isoprenaline to elevate cyclic AMP levels in intact cells expressing these constructs. 3 Treatment of cells expressing constitutively active mutant b 2 -adrenoceptor-Renilla luciferase with antagonist/inverse agonist ligands resulted in upregulation of levels of this polypeptide which could be monitored by the elevated luciferase activity. 4 The pEC 50 for ligand-induced luciferase upregulation and ligand a nity to bind the receptor were highly correlated. 5 Similar upregulation could be observed following sustained treatment with agonist ligands. 6 These e ects were only observed at a constitutively active mutant of the b 2 -adrenoceptor. Coexpression of the wild-type b 2 -adrenoceptor C-terminally tagged with the luciferase from Photinus pyralis did not result in ligand-induced upregulation of the levels of activity of this luciferase. 7 Co-expression of the constitutively active mutant b 2 -adrenoceptor-Renilla luciferase and an equivalent mutant of the a 1b -adrenoceptor C-terminally tagged with green¯uorescent protein allowed pharmacological selectivity of adrenoceptor antagonists to be demonstrated. 8 This approach o ers a sensitive and convenient means, which is amenable to high throughput analysis, to monitor ligand binding to a constitutively active mutant receptor. 9 As no prior knowledge of receptor ligands is required this approach may be suitable to identify ligands at orphan G protein-coupled receptors.
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