Selective Serotonin Reuptake Inhibitors within Cells: Temporal Resolution in Cytoplasm, Endoplasmic Reticulum, and Membrane.

Selective serotonin reuptake inhibitors (SSRIs) are the most prescribed treatment for individuals experiencing major depressive disorder. The therapeutic mechanisms that take place before, during, or after SSRIs bind the serotonin transporter (SERT) are poorly understood, partially because no studies exist on the cellular and subcellular pharmacokinetic properties of SSRIs in living cells. We studied escitalopram and fluoxetine using new intensity-based, drug-sensing fluorescent reporters targeted to the plasma membrane, cytoplasm, or endoplasmic reticulum (ER) of cultured neurons and mammalian cell lines. We also used chemical detection of drug within cells and phospholipid membranes. The drugs attain equilibrium in neuronal cytoplasm and ER at approximately the same concentration as the externally applied solution, with time constants of a few s (escitalopram) or 200-300 s (fluoxetine). Simultaneously, the drugs accumulate within lipid membranes by ≥18-fold (escitalopram) or 180-fold (fluoxetine), and possibly by much larger factors. Both drugs leave cytoplasm, lumen, and membranes just as quickly during washout. We synthesized membrane-impermeant quaternary amine derivatives of the two SSRIs. The quaternary derivatives are substantially excluded from membrane, cytoplasm, and ER for >2.4 h. They inhibit SERT transport-associated currents sixfold or 11-fold less potently than the SSRIs (escitalopram or fluoxetine derivative, respectively), providing useful probes for distinguishing compartmentalized SSRI effects. Although our measurements are orders of magnitude faster than the therapeutic lag of SSRIs, these data suggest that SSRI-SERT interactions within organelles or membranes may play roles during either the therapeutic effects or the antidepressant discontinuation syndrome.SIGNIFICANCE STATEMENT Selective serotonin reuptake inhibitors stabilize mood in several disorders. In general, these drugs bind to SERT, which clears serotonin from CNS and peripheral tissues. SERT ligands are effective and relatively safe; primary care practitioners often prescribe them. However, they have several side effects and require 2-6 weeks of continuous administration until they act effectively. How they work remains perplexing, contrasting with earlier assumptions that the therapeutic mechanism involves SERT inhibition followed by increased extracellular serotonin levels. This study establishes that two SERT ligands, fluoxetine and escitalopram, enter neurons within minutes, while simultaneously accumulating in many membranes. Such knowledge will motivate future research, hopefully revealing where and how SERT ligands engage their therapeutic target(s).

Three Mutations Convert the Selectivity of a Protein Sensor from Nicotinic Agonists to S-Methadone for Use in Cells, Organelles, and Biofluids.

We report a reagentless, intensity-based S-methadone fluorescent sensor, iS-methadoneSnFR, consisting of a circularly permuted GFP inserted within the sequence of a mutated bacterial periplasmic binding protein (PBP). We evolved a previously reported nicotine-binding PBP to become a selective S-methadone-binding sensor, via three mutations in the PBP's second shell and hinge regions. iS-methadoneSnFR displays the necessary sensitivity, kinetics, and selectivity─notably enantioselectivity against R-methadone─for biological applications. Robust iS-methadoneSnFR responses in human sweat and saliva and mouse serum enable diagnostic uses. Expression and imaging in mammalian cells demonstrate that S-methadone enters at least two organelles and undergoes acid trapping in the Golgi apparatus, where opioid receptors can signal. This work shows a straightforward strategy in adapting existing PBPs to serve real-time applications ranging from subcellular to personal pharmacokinetics.

Morality in minimally deceptive environments.

Psychologists, economists, and philosophers have long argued that in environments where deception is normative, moral behavior is harmed. In this article, we show that individuals making decisions within minimally deceptive environments do not behave more dishonestly than in nondeceptive environments. We demonstrate the latter using an example of experimental deception within established institutions, such as laboratories and institutional review boards. We experimentally manipulated whether participants received information about their deception. Across three well-powered studies, we empirically demonstrate that minimally deceptive environments do not affect downstream dishonest behavior. Only when participants were in a minimally deceptive environment and aware of being observed, their dishonest behavior decreased. Our results show that the relationship between deception and dishonesty might be more complicated than previous interpretations have suggested and expand the understanding of how deception might affect (im)moral behavior. We discuss possible limitations and future directions as well as the applied nature of these findings. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids.

We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for ΔF/F0 vs [nicotine] (δ-slope, 2.7 µM-1) is 6.1-fold higher than the previously reported iNicSnFR3a. The activated state of iNicSnFR12 has a fluorescence quantum yield of at least 0.6. We measured similar dose-response relations for the nicotine-induced absorbance increase and fluorescence increase, suggesting that the absorbance increase leads to the fluorescence increase via the previously described nicotine-induced conformational change, the "candle snuffer" mechanism. Molecular dynamics (MD) simulations identified a binding pose for nicotine, previously indeterminate from experimental data. MD simulations also showed that Helix 4 of the periplasmic binding protein (PBP) domain appears tilted in iNicSnFR12 relative to iNicSnFR3a, likely altering allosteric network(s) that link the ligand binding site to the fluorophore. In thermal melt experiments, nicotine stabilized the PBP of the tested iNicSnFR variants. iNicSnFR12 resolved nicotine in diluted mouse and human serum at 100 nM, the peak [nicotine] that occurs during smoking or vaping, and possibly at the decreasing levels during intervals between sessions. NicSnFR12 was also partially activated by unidentified endogenous ligand(s) in biofluids. Improved iNicSnFR12 variants could become the molecular sensors in continuous nicotine monitors for animal and human biofluids.

Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids.

We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for ∆F/F0vs [nicotine] (δ-slope, 2.7 µM-1) is 6.1-fold higher than the previously reported iNicSnFR3a. The activated state of iNicSnFR12 has a fluorescence quantum yield of at least 0.6. We measured similar dose-response relations for the nicotine-induced absorbance increase and fluorescence increase, suggesting that the absorbance increase leads to the fluorescence increase via the previously described nicotine-induced conformational change, the 'candle snuffer' mechanism. Molecular dynamics (MD) simulations identified a binding pose for nicotine, previously indeterminate from experimental data. MD simulations also showed that Helix 4 of the periplasmic binding protein (PBP) domain appears tilted in iNicSnFR12 relative to iNicSnFR3a, likely altering allosteric network(s) that link the ligand binding site to the fluorophore. In thermal melt experiments, nicotine stabilized the PBP of the tested iNicSnFR variants. iNicSnFR12 resolved nicotine in diluted mouse and human serum at 100 nM, the peak [nicotine] that occurs during smoking or vaping, and possibly at the decreasing levels during intervals between sessions. NicSnFR12 was also partially activated by unidentified endogenous ligand(s) in biofluids. Improved iNicSnFR12 variants could become the molecular sensors in continuous nicotine monitors for animal and human biofluids.

A field study of the impacts of workplace diversity on the recruitment of minority group members.

Increasing workplace diversity is a common goal. Given research showing that minority applicants anticipate better treatment in diverse workplaces, we ran a field experiment (N = 1,585 applicants, N = 31,928 website visitors) exploring how subtle organizational diversity cues affected applicant behaviour. Potential applicants viewed a company with varying levels of racial/ethnic or gender diversity. There was little evidence that racial/ethnic or gender diversity impacted the demographic composition or quality of the applicant pool. However, fewer applications were submitted to organizations with one form of diversity (that is, racial/ethnic or gender diversity), and more applications were submitted to organizations with only white men employees or employees diverse in race/ethnicity and gender. Finally, exploratory analyses found that female applicants were rated as more qualified than male applicants. Presenting a more diverse workforce does not guarantee more minority applicants, and organizations seeking to recruit minority applicants may need stronger displays of commitments to diversity.

Fluorescence Screens for Identifying Central Nervous System-Acting Drug-Biosensor Pairs for Subcellular and Supracellular Pharmacokinetics.

Subcellular pharmacokinetic measurements have informed the study of central nervous system (CNS)-acting drug mechanisms. Recent investigations have been enhanced by the use of genetically encoded fluorescent biosensors for drugs of interest at the plasma membrane and in organelles. We describe screening and validation protocols for identifying hit pairs comprising a drug and biosensor, with each screen including 13-18 candidate biosensors and 44-84 candidate drugs. After a favorable hit pair is identified and validated via these protocols, the biosensor is then optimized, as described in other papers, for sensitivity and selectivity to the drug. We also show sample hit pair data that may lead to future intensity-based drug-sensing fluorescent reporters (iDrugSnFRs). These protocols will assist scientists to use fluorescence responses as criteria in identifying favorable fluorescent biosensor variants for CNS-acting drugs that presently have no corresponding biosensor partner. This protocol was validated in: eLife (2022), DOI: 10.7554/eLife.74648 Graphical abstract.

Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands.

Nicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug-sensing fluorescent reporters (iDrugSnFRs) for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives - 10-fluorocytisine and 9-bromo-10-ethylcytisine. We report the first atomic-scale structures of liganded periplasmic binding protein-based biosensors, accelerating development of iDrugSnFRs and also explaining the activation mechanism. The nicotinic iDrugSnFRs detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by >30-fold. The new nicotinic iDrugSnFRs provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.

Replicating and extending the effects of auditory religious cues on dishonest behavior.

Although scientists agree that replications are critical to the debate on the validity of religious priming research, religious priming replications are scarce. This paper attempts to replicate and extend previously observed effects of religious priming on ethical behavior. We test the effect of religious instrumental music on individuals' ethical behavior with university participants (N = 408) in the Czech Republic, Japan, and the US. Participants were randomly assigned to listen to one of three musical tracks (religious, secular, or white noise) or to no music (control) for the duration of a decision-making game. Participants were asked to indicate which side of a vertically-bisected computer screen contained more dots and, in every trial, indicating that the right side of the screen had more dots earned participants the most money (irrespective of the number of dots). Therefore, participants were able to report dishonestly to earn more money. In agreement with previous research, we did not observe any main effects of condition. However, we were unable to replicate a moderating effect of self-reported religiosity on the effects of religious music on ethical behavior. Nevertheless, further analyses revealed moderating effects for ritual participation and declared religious affiliation congruent with the musical prime. That is, participants affiliated with a religious organization and taking part in rituals cheated significantly less than their peers when listening to religious music. We also observed significant differences in cheating behavior across samples. On average, US participants cheated the most and Czech participants cheated the least. We conclude that normative conduct is, in part, learned through active membership in religious communities and our findings provide further support for religious music as a subtle, moral cue.

Biosensors Show the Pharmacokinetics of S-Ketamine in the Endoplasmic Reticulum.

The target for the "rapid" (<24 h) antidepressant effects of S-ketamine is unknown, vitiating programs to rationally develop more effective rapid antidepressants. To describe a drug's target, one must first understand the compartments entered by the drug, at all levels-the organ, the cell, and the organelle. We have, therefore, developed molecular tools to measure the subcellular, organellar pharmacokinetics of S-ketamine. The tools are genetically encoded intensity-based S-ketamine-sensing fluorescent reporters, iSKetSnFR1 and iSKetSnFR2. In solution, these biosensors respond to S-ketamine with a sensitivity, S-slope = delta(F/F0)/(delta[S-ketamine]) of 0.23 and 1.9/µM, respectively. The iSKetSnFR2 construct allows measurements at <0.3 µM S-ketamine. The iSKetSnFR1 and iSKetSnFR2 biosensors display >100-fold selectivity over other ligands tested, including R-ketamine. We targeted each of the sensors to either the plasma membrane (PM) or the endoplasmic reticulum (ER). Measurements on these biosensors expressed in Neuro2a cells and in human dopaminergic neurons differentiated from induced pluripotent stem cells (iPSCs) show that S-ketamine enters the ER within a few seconds after appearing in the external solution near the PM, then leaves as rapidly after S-ketamine is removed from the extracellular solution. In cells, S-slopes for the ER and PM-targeted sensors differ by <2-fold, indicating that the ER [S-ketamine] is less than 2-fold different from the extracellular [S-ketamine]. Organelles represent potential compartments for the engagement of S-ketamine with its antidepressant target, and potential S-ketamine targets include organellar ion channels, receptors, and transporters.

Determining the pharmacokinetics of nicotinic drugs in the endoplasmic reticulum using biosensors.

Nicotine dependence is thought to arise in part because nicotine permeates into the endoplasmic reticulum (ER), where it binds to nicotinic receptors (nAChRs) and begins an "inside-out" pathway that leads to up-regulation of nAChRs on the plasma membrane. However, the dynamics of nicotine entry into the ER are unquantified. Here, we develop a family of genetically encoded fluorescent biosensors for nicotine, termed iNicSnFRs. The iNicSnFRs are fusions between two proteins: a circularly permutated GFP and a periplasmic choline-/betaine-binding protein engineered to bind nicotine. The biosensors iNicSnFR3a and iNicSnFR3b respond to nicotine by increasing fluorescence at [nicotine] <1 µM, the concentration in the plasma and cerebrospinal fluid of a smoker. We target iNicSnFR3 biosensors either to the plasma membrane or to the ER and measure nicotine kinetics in HeLa, SH-SY5Y, N2a, and HEK293 cell lines, as well as mouse hippocampal neurons and human stem cell-derived dopaminergic neurons. In all cell types, we find that nicotine equilibrates in the ER within 10 s (possibly within 1 s) of extracellular application and leaves as rapidly after removal from the extracellular solution. The [nicotine] in the ER is within twofold of the extracellular value. We use these data to run combined pharmacokinetic and pharmacodynamic simulations of human smoking. In the ER, the inside-out pathway begins when nicotine becomes a stabilizing pharmacological chaperone for some nAChR subtypes, even at concentrations as low as ∼10 nM. Such concentrations would persist during the 12 h of a typical smoker's day, continually activating the inside-out pathway by >75%. Reducing nicotine intake by 10-fold decreases activation to ∼20%. iNicSnFR3a and iNicSnFR3b also sense the smoking cessation drug varenicline, revealing that varenicline also permeates into the ER within seconds. Our iNicSnFRs enable optical subcellular pharmacokinetics for nicotine and varenicline during an early event in the inside-out pathway.

α1-FANGs: Protein Ligands Selective for the α-Bungarotoxin Site of the α1-Nicotinic Acetylcholine Receptor.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that play a central role in neuronal and neuromuscular signal transduction. Here, we have developed FANG ligands, fibronectin antibody-mimetic nicotinic acetylcholine receptor-generated ligands, using mRNA display. We generated a 1 trillion-member primary e10FnIII library to target a stabilized α1 nicotinic subunit (α211). This library yielded 270000 independent potential protein binding ligands. The lead sequence, α1-FANG1, represented 25% of all library sequences, showed the highest-affinity binding, and competed with α-bungarotoxin (α-Btx). To improve this clone, a new library based on α1-FANG1 was subjected to heat, protease, binding, off-rate selective pressures, and point mutations. This resulted in α1-FANG2 and α1-FANG3. These proteins bind α211 with KD values of 3.5 nM and 670 pM, respectively, compete with α-Btx, and show improved subunit specificity. α1-FANG3 is thermostable ( Tm = 62 °C) with a 6 kcal/mol improvement in folding free energy compared with that of the parent α1-FANG1. α1-FANG3 competes directly with the α-Btx binding site of intact neuromuscular heteropentamers [(α1)2ß1γδ] in mammalian culture-derived cellular membranes and in Xenopus laevis oocytes expressing these nAChRs. This work demonstrates that mRNA display against a monomeric ecto-domain of a pentamer has the capability to select ligands that bind that subunit in both a monomeric and a pentameric context. Overall, our work provides a route to creating a new family of stable, well-behaved proteins that specifically target this important receptor family.

Music As a Sacred Cue? Effects of Religious Music on Moral Behavior.

Religion can have an important influence in moral decision-making, and religious reminders may deter people from unethical behavior. Previous research indicated that religious contexts may increase prosocial behavior and reduce cheating. However, the perceptual-behavioral link between religious contexts and decision-making lacks thorough scientific understanding. This study adds to the current literature by testing the effects of purely audial religious symbols (instrumental music) on moral behavior across three different sites: Mauritius, the Czech Republic, and the USA. Participants were exposed to one of three kinds of auditory stimuli (religious, secular, or white noise), and subsequently were given a chance to dishonestly report on solved mathematical equations in order to increase their monetary reward. The results showed cross-cultural differences in the effects of religious music on moral behavior, as well as a significant interaction between condition and religiosity across all sites, suggesting that religious participants were more influenced by the auditory religious stimuli than non-religious participants. We propose that religious music can function as a subtle cue associated with moral standards via cultural socialization and ritual participation. Such associative learning can charge music with specific meanings and create sacred cues that influence normative behavior. Our findings provide preliminary support for this view, which we hope further research will investigate more closely.

An evaluation of Poisson-Boltzmann electrostatic free energy calculations through comparison with experimental mutagenesis data.

For systems involving highly and oppositely charged proteins, electrostatic forces dominate association and contribute to biomolecular complex stability. Using experimental or theoretical alanine-scanning mutagenesis, it is possible to elucidate the contribution of individual ionizable amino acids to protein association. We evaluated our electrostatic free energy calculations by comparing calculated and experimental data for alanine mutants of five protein complexes. We calculated Poisson-Boltzmann electrostatic free energies based on a thermodynamic cycle, which incorporates association in a reference (Coulombic) and solvated (solution) state, as well as solvation effects. We observe that Coulombic and solvation free energy values correlate with experimental data in highly and oppositely charged systems, but not in systems comprised of similarly charged proteins. We also observe that correlation between solution and experimental free energies is dependent on dielectric coefficient selection for the protein interior. Free energy correlations improve as protein dielectric coefficient increases, suggesting that the protein interior experiences moderate dielectric screening, despite being shielded from solvent. We propose that higher dielectric coefficients may be necessary to more accurately predict protein-protein association. Additionally, our data suggest that Coulombic potential calculations alone may be sufficient to predict relative binding of protein mutants.