A Reckless Guide to P-values : Local Evidence, Global Errors.

This chapter demystifies P-values, hypothesis tests and significance tests and introduces the concepts of local evidence and global error rates. The local evidence is embodied in this data and concerns the hypotheses of interest for this experiment, whereas the global error rate is a property of the statistical analysis and sampling procedure. It is shown using simple examples that local evidence and global error rates can be, and should be, considered together when making inferences. Power analysis for experimental design for hypothesis testing is explained, along with the more locally focussed expected P-values. Issues relating to multiple testing, HARKing and P-hacking are explained, and it is shown that, in many situations, their effects on local evidence and global error rates are in conflict, a conflict that can always be overcome by a fresh dataset from replication of key experiments. Statistics is complicated, and so is science. There is no singular right way to do either, and universally acceptable compromises may not exist. Statistics offers a wide array of tools for assisting with scientific inference by calibrating uncertainty, but statistical inference is not a substitute for scientific inference. P-values are useful indices of evidence and deserve their place in the statistical toolbox of basic pharmacologists.

Cortisol limits selected actions of synthetic glucocorticoids in the airway epithelium.

Cortisol, a physiologic glucocorticoid (GC), is essential for growth and differentiation of the airway epithelium. Epithelial function influences inflammation in chronic respiratory diseases. Synthetic GCs, including inhaled corticosteroids, exert anti-inflammatory effects in airway epithelium by transactivation of genes and by inhibition of proinflammatory cytokine release. We examined the effect of cortisol on the actions of synthetic GCs in the airway epithelium, demonstrating that cortisol acts like a partial agonist at the GC receptor (GR), limiting GC-induced GR-dependent transcription in the BEAS-2B human bronchial epithelial cell line. Cortisol also limited the inhibition of granulocyte macrophage colony-stimulating factor release by synthetic GCs in TNF-α-activated BEAS-2B cells. The relevance of these findings is supported by observations on tracheal epithelium obtained from mice treated for 5 d with systemic GC, showing limitations in selected GC effects, including inhibition of IL-6. Moreover, gene transactivation by synthetic GCs was compromised by standard air-liquid interface (ALI) growth medium cortisol concentration (1.4 µM) in the ALI-differentiated organotypic culture of primary human airway epithelial cells. These findings suggest that endogenous corticosteroids may limit certain actions of synthetic pharmacological GCs and contribute to GC insensitivity, particularly when corticosteroid levels are elevated by stress.-Prodanovic, D., Keenan, C. R., Langenbach, S., Li, M., Chen, Q., Lew, M. J., Stewart, A. G. Cortisol limits selected actions of synthetic glucocorticoids in the airway epithelium.

Protease-activated receptor 2 (PAR2) protein and transient receptor potential vanilloid 4 (TRPV4) protein coupling is required for sustained inflammatory signaling.

G protein-coupled receptors of nociceptive neurons can sensitize transient receptor potential (TRP) ion channels, which amplify neurogenic inflammation and pain. Protease-activated receptor 2 (PAR(2)), a receptor for inflammatory proteases, is a major mediator of neurogenic inflammation and pain. We investigated the signaling mechanisms by which PAR(2) regulates TRPV4 and determined the importance of tyrosine phosphorylation in this process. Human TRPV4 was expressed in HEK293 cells under control of a tetracycline-inducible promoter, allowing controlled and graded channel expression. In cells lacking TRPV4, the PAR(2) agonist stimulated a transient increase in [Ca(2+)](i). TRPV4 expression led to a markedly sustained increase in [Ca(2+)](i). Removal of extracellular Ca(2+) and treatment with the TRPV4 antagonists Ruthenium Red or HC067047 prevented the sustained response. Inhibitors of phospholipase A(2) and cytochrome P450 epoxygenase attenuated the sustained response, suggesting that PAR(2) generates arachidonic acid-derived lipid mediators, such as 5',6'-EET, that activate TRPV4. Src inhibitor 1 suppressed PAR(2)-induced activation of TRPV4, indicating the importance of tyrosine phosphorylation. The TRPV4 tyrosine mutants Y110F, Y805F, and Y110F/Y805F were expressed normally at the cell surface. However, PAR(2) was unable to activate TRPV4 with the Y110F mutation. TRPV4 antagonism suppressed PAR(2) signaling to primary nociceptive neurons, and TRPV4 deletion attenuated PAR(2)-stimulated neurogenic inflammation. Thus, PAR(2) activation generates a signal that induces sustained activation of TRPV4, which requires a key tyrosine residue (TRPV4-Tyr-110). This mechanism partly mediates the proinflammatory actions of PAR(2).

N-glycosylation determines ionic permeability and desensitization of the TRPV1 capsaicin receptor.

The balance of glycosylation and deglycosylation of ion channels can markedly influence their function and regulation. However, the functional importance of glycosylation of the TRPV1 receptor, a key sensor of pain-sensing nerves, is not well understood, and whether TRPV1 is glycosylated in neurons is unclear. We report that TRPV1 is N-glycosylated and that N-glycosylation is a major determinant of capsaicin-evoked desensitization and ionic permeability. Both N-glycosylated and unglycosylated TRPV1 was detected in extracts of peripheral sensory nerves by Western blotting. TRPV1 expressed in HEK-293 cells exhibited various degrees of glycosylation. A mutant of asparagine 604 (N604T) was not glycosylated but did not alter plasma membrane expression of TRPV1. Capsaicin-evoked increases in intracellular calcium ([Ca(2+)](i)) were sustained in wild-type TRPV1 HEK-293 cells but were rapidly desensitized in N604T TRPV1 cells. There was marked cell-to-cell variability in capsaicin responses and desensitization between individual cells expressing wild-type TRPV1 but highly uniform responses in cells expressing N604T TRPV1, consistent with variable levels of glycosylation of the wild-type channel. These differences were also apparent when wild-type or N604T TRPV1-GFP fusion proteins were expressed in neurons from trpv1(-/-) mice. Capsaicin evoked a marked, concentration-dependent increase in uptake of the large cationic dye YO-PRO-1 in cells expressing wild-type TRPV1, indicative of loss of ion selectivity, that was completely absent in cells expressing N604T TRPV1. Thus, TRPV1 is variably N-glycosylated and glycosylation is a key determinant of capsaicin regulation of TRPV1 desensitization and permeability. Our findings suggest that physiological or pathological alterations in TRPV1 glycosylation would affect TRPV1 function and pain transmission.

Ligand-supported purification of the urotensin-II receptor.

A crucial limitation for structural and biophysical analysis of G protein-coupled receptors (GPCRs) is the inherent challenge of purifying and stabilizing these receptors in an active (agonist-bound) conformation. Peptide ligands, such as the vasoactive, cyclic hormone urotensin-II (U-II), may provide new purification tools, via high affinity, pseudo-irreversible binding suitable for ligand-based affinity purification. We show that the U-II receptor (UT) is resistant to desensitization as a result of low phosphorylation and diminished endocytosis. UT also displays an unusual proclivity to remain active with vasoconstriction sustained despite extensive washout of the ligand. To exploit these properties for ligand-supported purification, we modified the U-II ligand by attaching a biotin moiety and spacer arm to the N terminus, creating a novel affinity ligand (Bio-U-II) to interface with streptavidin media. Bio-U-II bound to UT with pharmacological properties analogous to those of the unmodified U-II ligand (high-affinity, pseudo-irreversible binding). The prebinding of Bio-U-II to UT (before exposure to detergent) facilitated specific capture of UT by stabilizing the receptor structure during solubilization with detergent. Solubilization of UT with the most compatible detergent, n-dodecyl ß-d-maltoside, was dependent on the critical micelle concentration, and Gα(q/11) protein was copurified with captured Bio-U-II-UT complexes. Furthermore, captured Bio-U-II-UT complexes were resistant to dissociation at elevated temperatures, suggesting that UT is relatively thermostable, making it an ideal candidate for future structural and biophysical studies. This work demonstrates the utility of pseudo-irreversible ligands to support the purification of a GPCR during detergent extraction, resulting in the first successful purification of the UT.

Multi-Component Mechanism of H2 Relaxin Binding to RXFP1 through NanoBRET Kinetic Analysis.

The peptide hormone H2 relaxin has demonstrated promise as a therapeutic, but mimetic development has been hindered by the poorly understood relaxin receptor RXFP1 activation mechanism. H2 relaxin is hypothesized to bind to two distinct ECD sites, which reorientates the N-terminal LDLa module to activate the transmembrane domain. Here we provide evidence for this model in live cells by measuring bioluminescence resonance energy transfer (BRET) between nanoluciferase-tagged RXFP1 constructs and fluorescently labeled H2 relaxin (NanoBRET). Additionally, we validate these results using the related RXFP2 receptor and chimeras with an inserted RXFP1-binding domain utilizing NanoBRET and nuclear magnetic resonance studies on recombinant proteins. We therefore provide evidence for the multi-component molecular mechanism of H2 relaxin binding to RXFP1 on the full-length receptor in cells. Also, we show the utility of NanoBRET real-time binding kinetics to reveal subtle binding complexities, which may be overlooked in traditional equilibrium binding assays.

Principles: when there should be no difference--how to fail to reject the null hypothesis.

It is common to perform experiments in which a 'success' is claimed when the null hypothesis is discarded. However, there is a category of experiment that has become important in which a success is when the null hypothesis is not rejected. Failing to discard the null hypothesis is different from proving it to be valid, a distinction that is particularly important in experiments in which any inadequacy of experimental design or implementation enhances the likelihood of a success. The appropriate analysis of such experiments tests for evidence of the validity of the null hypothesis rather than simply failing to find evidence against it.

Biased signalling from the glucocorticoid receptor: Renewed opportunity for tailoring glucocorticoid activity.

Recent landmark studies applying analytical pharmacology approaches to the glucocorticoid receptor (GR) have demonstrated that different ligands can cause differential activation of distinct GR-regulated genes. Drawing on concepts of signalling bias from the field of G protein-coupled receptor (GPCR) biology, we speculate that ligand-dependent differences in GR signalling can be considered analogous to GPCR biased signalling, and thus can be quantitatively analysed in a similar way. This type of approach opens up the possibility of using rational structure-based drug optimisation strategies to improve the therapeutic selectivity of glucocorticoid drugs to maximise their efficacy and minimise adverse effects.

The two-state model of antagonist-AT1 receptor interaction: an hypothesis defended but not tested.

A correlation between insurmountable antagonism and slow dissociation has been observed for the non-peptidic AT1 receptor antagonists. This commentary examines the validity of conclusions regarding a two stage binding mechanism that has been proposed in order to account for both the slow dissociation and insurmountable antagonism. Support for that hypothetical mechanism is in the form of the goodness of fit between experimental data and modelled data in a number of papers from the same laboratory. We challenge the idea that a simple match of model and data is an adequate test of an hypothesis by showing that a simpler model matches the data equally well. We conclude that two stage binding is not necessary to explain the behaviour of AT1 receptor antagonists.

Bad statistical practice in pharmacology (and other basic biomedical disciplines): you probably don't know P.

Statistical analysis is universally used in the interpretation of the results of basic biomedical research, being expected by referees and readers alike. Its role in helping researchers to make reliable inference from their work and its contribution to the scientific process cannot be doubted, but can be improved. There is a widespread and pervasive misunderstanding of P-values that limits their utility as a guide to inference, and a change in the manner in which P-values are specified and interpreted will lead to improved outcomes. This paper explains the distinction between Fisher's P-values, which are local indices of evidence against the null hypothesis in the results of a particular experiment, and Neyman-Pearson α levels, which are global rates of false positive errors from unrelated experiments taken as an aggregate. The vast majority of papers published in pharmacological journals specify P-values, either as exact-values or as being less than a value (usually 0.05), but they are interpreted in a hybrid manner that detracts from their Fisherian role as indices of evidence without gaining the control of false positive and false negative error rate offered by a strict Neyman-Pearson approach. An informed choice between those approaches offers substantial advantages to the users of statistical tests over the current accidental hybrid approach.

Estimating the risk of rare complications: is the 'rule of three' good enough?

THE CLINICAL PROBLEM: If a surgeon has performed a particular operation on n consecutive patients without major complications, what is the long-term risk of major complications after performing many more such operations? Examples of such operations are endoscopic cholecystectomy, nephrectomy and sympathectomy. THE STATISTICAL PROBLEM AND SOLUTIONS: This general problem has exercised the minds of theoretical statisticians for more than 80 years. They agree only that the long-term risk is best expressed as the upper bound of a 95% confidence interval. We consider many proposed solutions, from those that involve complex statistical theory to the empirical 'rule of three', popular among clinicians, in which the percentage risk is given by the formula 100 x (3/n). OUR CONCLUSIONS: The 'rule of three' grossly underestimates the future risks and can be applied only when the initial complication rate is zero (that is, 0/n). If the initial complication rate is greater than zero, then no simple 'rule' suffices. We give the results of applying the more popular statistical models, including their coverage. The 'exact' Clopper-Pearson interval has wider coverage across all proportions than its nominal 95%, and is, thus, too conservative. The Wilson score confidence interval gives about 95% coverage on average overall population proportions, except very small ones, so we prefer it to the Clopper-Pearson method. Unlike all the other intervals, Bayesian intervals with uniform priors yield exactly 95% coverage at any observed proportion. Thus, we strongly recommend Bayesian intervals and provide free software for executing them.

A novel mechanism of G protein-coupled receptor functional selectivity. Muscarinic partial agonist McN-A-343 as a bitopic orthosteric/allosteric ligand.

Many G protein-coupled receptors (GPCRs) possess allosteric binding sites distinct from the orthosteric site utilized by their cognate ligands, but most GPCR allosteric modulators reported to date lack signaling efficacy in their own right. McN-A-343 (4-(N-(3-chlorophenyl)carbamoyloxy)-2-butynyltrimethylammonium chloride) is a functionally selective muscarinic acetylcholine receptor (mAChR) partial agonist that can also interact allosterically at the M(2) mAChR. We hypothesized that this molecule simultaneously utilizes both an allosteric and the orthosteric site on the M(2) mAChR to mediate these effects. By synthesizing progressively truncated McN-A-343 derivatives, we identified two, which minimally contain 3-chlorophenylcarbamate, as pure allosteric modulators. These compounds were positive modulators of the orthosteric antagonist N-[(3)H]methylscopolamine, but in functional assays of M(2) mAChR-mediated ERK1/2 phosphorylation and guanosine 5'-3-O-([(35)S]thio)triphosphate binding, they were negative modulators of agonist efficacy. This negative allosteric effect was diminished upon mutation of Y177A in the second extracellular (E2) loop of the M(2) mAChR that is known to reduce prototypical allosteric modulator potency. Our results are consistent with McN-A-343 being a bitopic orthosteric/allosteric ligand with the allosteric moiety engendering partial agonism and functional selectivity. This finding suggests a novel and largely unappreciated mechanism of "directed efficacy" whereby functional selectivity may be engendered in a GPCR by utilizing an allosteric ligand to direct the signaling of an orthosteric ligand encoded within the same molecule.

Side-chain substitutions within angiotensin II reveal different requirements for signaling, internalization, and phosphorylation of type 1A angiotensin receptors.

Binding of the peptide hormone angiotensin II (AngII) to the type 1 (AT(1A)) receptor and the subsequent activation of phospholipase C-mediated signaling, involves specific determinants within the AngII peptide sequence. In contrast, the contribution of such determinants to AT(1A) receptor internalization, phosphorylation and activation of mitogen-activated protein kinase (MAPK) signaling is not known. In this study, the internalization of an enhanced green fluorescent protein-tagged AT(1A) receptor (AT(1A)-EGFP), in response to AngII and a series of substituted analogs, was visualized and quantified using confocal microscopy. AngII-stimulation resulted in a rapid, concentration-dependent internalization of the chimeric receptor, which was prevented by pretreatment with the nonpeptide AT(1) receptor antagonist EXP3174. Remarkably, AT(1A) receptor internalization was unaffected by substitution of AngII side chains, including single and double substitutions of Tyr(4) and Phe(8) that abolish phospholipase C signaling through the receptor. AngII-induced receptor phosphorylation was significantly inhibited by several substitutions at Phe(8) as well as alanine replacement of Asp(1). The activation of MAPK was only significantly inhibited by substitutions at position eight in the peptide and specific substitutions did not equally inhibit inositol phosphate production, receptor phosphorylation and MAPK activation. These results indicate that separate, yet overlapping, contacts made between the AngII peptide and the AT(1A) receptor select/induce distinct receptor conformations that preferentially affect particular receptor outcomes. The requirements for AT(1A) receptor internalization seem to be less stringent than receptor activation and signaling, suggesting an inherent bias toward receptor deactivation.