Microsecond Folding of preQ1 Riboswitch and Its Biological Significance Revealed by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy.

Riboswitches are regulatory elements of bacterial mRNA which function with conformational switching upon binding of specific cellular metabolites. In particular, transcriptional riboswitches regulate gene expression kinetically through the conformational change of the aptamer domain. In this study, we investigate the conformational dynamics and ligand binding mechanisms of the aptamer domain of a transcriptional prequeuosine (preQ1) riboswitch from Bacillus subtilis using two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS) with microsecond time resolution. The obtained time-resolved single-molecule data indicate that the aptamer domain undergoes folding/unfolding including three forms, which are attributed to hairpin (O), pseudoknot-like (pF), and H-type pseudoknot (fF) structures. It is found that a cofactor, Mg2+, binds only to the fF form with the conformational selection mechanism. In contrast, it is indicated that the ligand, preQ1, binds to the O form with the induced-fit mechanism and significantly accelerates the microsecond O → pF folding process. It is also shown that the binding with preQ1 substantially stabilizes the fF form that is generated from the pF form with a long time constant (>10 ms). Combining these results with the results of a former smFRET study on the slower time scale, we obtain an overall picture of the folding/unfolding dynamics of the aptamer domain as well as its energy landscape. On the basis of the picture obtained, we discuss the significance of the microsecond folding/unfolding of the aptamer domain for biological function of the riboswitch and propose the molecular mechanism of the gene expression controlled by the structural dynamics of the aptamer domain.

India's response to adolescent mental health: a policy review and stakeholder analysis.

PURPOSE: Mental health problems and suicide are the leading cause of mortality in young people globally. India is home to the largest number of adolescents in the world. This study was undertaken to assess the policy environment for addressing adolescent mental health in India. METHODS: We conducted a review of 6 policies and programs and 11 in-depth interviews with key stakeholders. The findings were analyzed using the policy triangle analysis framework (i.e., context, content, actors and process). RESULTS: There is no conformity of the age ranges addressed by these documents nor are vulnerable groups explicitly recognized. Stress, anxiety and depression were commonly identified as mental health concerns and diverse platforms such as community, family, school, digital and health facility were recommended to deliver preventive and treatment interventions. Some interventions specifically targeted some social determinants (like safe and supportive schools) but many others (like social norms) were not addressed. Preventive interventions were recommended for delivery through peers and other non-specialist providers while treatment interventions were recommended for delivery in healthcare facilities by specialist health professionals. There was very little engagement of young people in the development of these policies or in their implementation, except for peer educators mentioned in one policy. Stakeholders identified several major challenges in implementing these policies, notably the lack of inter-sectoral coordination and fragmentation of governance; budgetary constraints; and scanty human resources. CONCLUSIONS: Although there are now several policy instruments testifying to a comprehensive approach on adolescent mental health, there are gaps in the extent of engagement of young people and how these will be operationalized that may limit their impact on addressing the burden of mental health problems in young people in India.

Effectiveness of a brief community outreach tobacco cessation intervention in India: a cluster-randomised controlled trial (the BABEX Trial).

BACKGROUND: Tobacco use kills half a million people every month, most in low-middle income countries (LMICs). There is an urgent need to identify potentially low-cost, scalable tobacco cessation interventions for these countries. OBJECTIVE: To evaluate a brief community outreach intervention delivered by health workers to promote tobacco cessation in India. DESIGN: Cluster-randomised controlled trial. SETTING: 32 low-income administrative blocks in Delhi, half government authorised ('resettlement colony') and half unauthorised ('J.J. cluster') communities. PARTICIPANTS: 1213 adult tobacco users. INTERVENTIONS: Administrative blocks were computer randomised in a 1:1 ratio, to the intervention (16 clusters; n=611) or control treatment (16 clusters; n=602), delivered and assessed at individual level between 07/2012 and 11/2013. The intervention was single session quit advice (15 min) plus a single training session in yogic breathing exercises; the control condition comprised very brief quit advice (1 min) alone. Both were delivered via outreach, with contact made though household visits. MEASUREMENTS: The primary outcome was 6-month sustained abstinence from all tobacco, assessed 7 months post intervention delivery, biochemically verified with salivary cotinine. RESULTS: The smoking cessation rate was higher in the intervention group (2.6% (16/611)) than in the control group (0.5% (3/602)) (relative risk=5.32, 95% CI 1.43 to 19.74, p=0.013). There was no interaction with type of tobacco use (smoked vs smokeless). Results did not change materially in adjusted analyses, controlling for participant characteristics. CONCLUSIONS: A single session community outreach intervention can increase tobacco cessation in LMIC. The effect size, while small, could impact public health if scaled up with high coverage. TRIAL REGISTRATION NUMBER: ISRCTCN23362894.

The Social Gradient in Tobacco Use Does Not Generalize to Low-Income Urban Communities in India: Findings From a Census Survey.

INTRODUCTION: The existence of a social gradient in tobacco use has been clearly established in a number of countries with people with lower socioeconomic status being more likely to use tobacco. It is not clear how far this gradient is evident within severely deprived communities. This study assessed the association between occupation as a marker of socioeconomic status and use of smoked and smokeless tobacco within "slum" areas of Delhi, India. METHODS: A census survey of 11 888 households, comprising 30 655 adults from 28 low-income communities (14 government-authorized and 14 unauthorized settlements called "Jhuggi-Jhopri/JJ" clusters) was conducted in 2012. The survey assessed age, sex, household size, occupational group, and current tobacco use. Independent associations with tobacco use were conducted using complex samples regression analysis, stratified by gender. RESULTS: A quarter of participants (24.3%, 95% confidence interval [CI] 21.5-27.5) used any tobacco. Slightly more people used smoked (14.6%, 95% CI 12.9-16.3) than smokeless (12.6%, 95% CI 10.7-14.8) tobacco, with a small minority being dual users (2.7%, 95% CI 2.1-3.5). Prevalence of any tobacco use was highest in unskilled (45.13%, 95% CI 42.4-47.9) and skilled (46.2%, 95% CI 41.1-51.4) manual occupations and lower in nonmanual (30.3%, 95% CI 26.2-34.7) occupations and those who were unemployed (29.0%, 95% CI 25.3-33.0). This was confirmed in adjusted analysis in men but associations were more complex in women. CONCLUSIONS: Use of smoked and smokeless tobacco in low-income urban communities in India has a complex association with occupational status with both nonmanual occupation and unemployment being associated with lower prevalence of smoked and smokeless tobacco in men. IMPLICATIONS: Tobacco use in high-income countries shows a strong inverse relationship with social grade, income, and deprivation such that use is much more common among those who can least afford it. This study is the first to look at this social gradient in the context of low-income communities in India, finding that both unemployment and nonmanual occupation were associated with lower rates of tobacco use in men. The data present a challenge to existing explanations of the social gradient, requiring further consideration of the conditions under which affordability may work to reduce health inequalities arising from tobacco use.

Steric Crowding of the Turn Region Alters the Tertiary Fold of Amyloid-ß18-35 and Makes It Soluble.

Aß self-assembles into parallel cross-ß fibrillar aggregates, which is associated with Alzheimer's disease pathology. A central hairpin turn around residues 23-29 is a defining characteristic of Aß in its aggregated state. Major biophysical properties of Aß, including this turn, remain unaltered in the central fragment Aß18-35. Here, we synthesize a single deletion mutant, ΔG25, with the aim of sterically hindering the hairpin turn in Aß18-35. We find that the solubility of the peptide goes up by more than 20-fold. Although some oligomeric structures do form, solution state NMR spectroscopy shows that they have mostly random coil conformations. Fibrils ultimately form at a much higher concentration but have widths approximately twice that of Aß18-35, suggesting an opening of the hairpin bend. Surprisingly, two-dimensional solid state NMR shows that the contact between Phe(19) and Leu(34) residues, observed in full-length Aß and Aß18-35, is still intact in these fibrils. This is possible if the monomers in the fibril are arranged in an antiparallel ß-sheet conformation. Indeed, IR measurements, supported by tyrosine cross-linking experiments, provide a characteristic signature of the antiparallel ß-sheet. We conclude that the self-assembly of Aß is critically dependent on the hairpin turn and on the contact between the Phe(19) and Leu(34) regions, making them potentially sensitive targets for Alzheimer's therapeutics. Our results show the importance of specific conformations in an aggregation process thought to be primarily driven by nonspecific hydrophobic interactions.

Curcumin alters the salt bridge-containing turn region in amyloid ß(1-42) aggregates.

Amyloid ß (Aß) fibrillar deposits in the brain are a hallmark of Alzheimer disease (AD). Curcumin, a common ingredient of Asian spices, is known to disrupt Aß fibril formation and to reduce AD pathology in mouse models. Understanding the structural changes induced by curcumin can potentially lead to AD pharmaceutical agents with inherent bio-compatibility. Here, we use solid-state NMR spectroscopy to investigate the structural modifications of amyloid ß(1-42) (Aß42) aggregates induced by curcumin. We find that curcumin induces major structural changes in the Asp-23-Lys-28 salt bridge region and near the C terminus. Electron microscopy shows that the Aß42 fibrils are disrupted by curcumin. Surprisingly, some of these alterations are similar to those reported for Zn(2+) ions, another agent known to disrupt the fibrils and alter Aß42 toxicity. Our results suggest the existence of a structurally related family of quasi-fibrillar conformers of Aß42, which is stabilized both by curcumin and by Zn(2+.)

An acute flaccid paralysis surveillance-based serosurvey of poliovirus antibodies in Western Uttar Pradesh, India.

BACKGROUND: Despite intensified use of monovalent oral poliovirus type 1 vaccine and improved coverage of immunization campaigns, wild poliovirus type 1 persisted in Indian states of Uttar Pradesh and Bihar during 2006 to 2009. METHODS: A serosurvey was conducted among cases of acute flaccid paralysis in the 25 high-polio-incidence districts of western Uttar Pradesh. Children were recruited by age group (6-11 months, 12-24 months, and 25-69 months) from among cases reported through the acute flaccid paralysis surveillance system between November 2008 and August 2009. RESULTS: Seroprevalence for type 1 wild poliovirus was >96.4% for each age group. The seroprevalence of wild poliovirus types 2 and 3 increased with age, from 36.7% to 73.4% for type 2 and from 39.0% to 74.1% for type 3. In addition to the number of type-specific vaccine doses, father's level of education, being from a Muslim family, height for age, and female sex were the socioeconomic risk factors associated with seronegativity to poliovirus. CONCLUSIONS: The seroprevalence and risk factors identified in this study were consistent with the epidemiology of polio, and the findings were instrumental in optimizing vaccination strategy in western Uttar Pradesh with respect to the choice of OPV types, the frequency of supplementary immunization campaigns, and the urgency to improve routine immunization services.

Assessing population immunity in a persistently high-risk area for wild poliovirus transmission in India: a serological study in Moradabad, Western Uttar Pradesh.

BACKGROUND: Moradabad district in Uttar Pradesh reported the highest number of paralytic polio cases in India during 2001-2007. We conducted a study in Moradabad in 2007 to assess seroprevalence against poliovirus types 1, 2, and 3 in children 6-12 and 36-59 months of age to guide future strategies to interrupt wild poliovirus transmission in high-risk areas. METHODS: Children attending 10 health facilities for minor illnesses who met criteria for study inclusion were eligible for enrollment. We recorded vaccination history, weight, and length and tested sera for neutralizing antibodies to poliovirus types 1, 2, and 3. RESULTS: Poliovirus type 1, 2, and 3 seroprevalences were 88% (95% confidence interval [CI], 84%-91%), 70% (95% CI, 66%-75%), and 75% (95% CI, 71%-79%), respectively, among 467 in the younger age group (n=467), compared with 100% (95% CI, 99%-100%), 97% (95% CI, 95%-98%), and 93% (91%-95%), respectively, among 447 children in the older age group (P<.001 for all serotypes). CONCLUSIONS: This seroprevalence study provided extremely useful information that was used by the program in India to guide immunization policies, such as optimizing the use of different OPV formulations in vaccination campaigns and strengthening routine immunization services. Similar surveys in populations at risk should be performed at regular intervals in countries where the risk of persistence or spread of indigenous or imported wild poliovirus is high.

Cross-sectional serologic assessment of immunity to poliovirus infection in high-risk areas of northern India.

INTRODUCTION: The objectives of this survey were to assess the seroprevalence of antibodies to poliovirus types 1 and 3 and the impact of bivalent (types 1 and 3) oral poliovirus vaccine (bOPV) use in immunization campaigns in northern India. METHODS: In August 2010, a 2-stage stratified cluster sampling method identified infants aged 6-7 months in high-risk blocks for wild poliovirus infection. Vaccination history, weight and length, and serum were collected to test for neutralizing antibodies to poliovirus types 1, 2, and 3. RESULTS: Seroprevalences of antibodies to poliovirus types 1, 2, and 3 were 98% (95% confidence interval [CI], 97%-99%), 66% (95% CI, 62%-69%), and 77% (95% CI, 75%-79%), respectively, among 664 infants from Bihar and 616 infants from Uttar Pradesh. Infants had received a median of 3 bOPV doses and 2 monovalent type 1 OPV (mOPV1) doses through campaigns and 3 trivalent OPV (tOPV) doses through routine immunization. Among subjects with 0 tOPV doses, the seroprevalences of antibodies to type 3 were 50%, 77%, and 82% after 2, 3, and 4 bOPV doses, respectively. In multivariable analysis, malnutrition was associated with a lower seroprevalence of type 3 antibodies. CONCLUSIONS: This study confirmed that replacing mOPV1 with bOPV in campaigns was successful in maintaining very high population immunity to type 1 poliovirus and substantially decreasing the immunity gap to type 3 poliovirus.

Significant structural differences between transient amyloid-ß oligomers and less-toxic fibrils in regions known to harbor familial Alzheimer's mutations.

Small oligomers of the amyloidâ€…ß (Aß) peptide, rather than the monomers or the fibrils, are suspected to initiate Alzheimer's disease (AD). However, their low concentration and transient nature under physiological conditions have made structural investigations difficult. A method for addressing such problems has been developed by combining rapid fluorescence techniques with slower two-dimensional solid-state NMR methods. The smallest Aß40 oligomers that demonstrate a potential sign of toxicity, namely, an enhanced affinity for cell membranes, were thus probed. The two hydrophobic regions (residues 10-21 and 30-40) have already attained the conformation that is observed in the fibrils. However, the turn region (residues 22-29) and the N-terminal tail (residues 1-9) are strikingly different. Notably, ten of eleven known Aß mutants that are linked to familial AD map to these two regions. Our results provide potential structural cues for AD therapeutics and also suggest a general method for determining transient protein structures.

Pulsed-Interleaved-Excitation Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy.

We report on pulsed-interleaved-excitation two-dimensional fluorescence lifetime correlation spectroscopy (PIE 2D FLCS) to study biomolecular structural dynamics with high sensitivity and high time resolution using Förster resonance energy transfer (FRET). PIE 2D FLCS is an extension of 2D FLCS, which is a unique single-molecule fluorescence method that uses fluorescence lifetime information to distinguish different fluorescence species in equilibrium and resolves their interconversion dynamics with a submicrosecond time resolution. Because 2D FLCS has used only a single-color excitation so far, it was difficult to distinguish a very low-FRET (or zero-FRET) species from only donor-labeled species. We overcome this difficulty by implementing the PIE scheme (i.e., alternate excitation of the donor and acceptor dyes using two temporally interleaved excitations with different colors) to 2D FLCS, realizing two-color excitation and two-color fluorescence detection in 2D FLCS. After proof-of-principle PIE 2D FLCS analysis on the photon data synthesized with Monte Carlo simulation, we apply PIE 2D FLCS to a DNA-hairpin sample and show that this method readily distinguishes four fluorescent species, i.e., high-FRET, low-FRET, and two single-dye-labeled species. In addition, we show that PIE 2D FLCS can also quantitatively evaluate the contributions of the donor-acceptor spectral crosstalk, which often appears as artifacts in FRET studies and degrades the information obtained.

A Toxicogenic Interaction between Intracellular Amyloid-ß and Apolipoprotein-E.

Alzheimer's disease (AD) is associated with the aggregation of amyloid ß (Aß) and tau proteins. Why ApoE variants are significant genetic risk factors remains a major unsolved puzzle in understanding AD, although intracellular interactions with ApoE are suspected to play a role. Here, we show that specific changes in the fluorescence lifetime of fluorescently tagged small Aß oligomers in rat brain cells correlate with the cellular ApoE content. An inhibitor of the Aß-ApoE interaction suppresses these changes and concomitantly reduces Aß toxicity in a dose-dependent manner. Single-molecule techniques show changes both in the conformation and in the stoichiometry of the oligomers. Neural stem cells derived from hiPSCs of Alzheimer's patients also exhibit these fluorescence lifetime changes. We infer that intracellular interaction with ApoE modifies the N-terminus of the Aß oligomers, inducing changes in their stoichiometry, membrane affinity, and toxicity. These changes can be directly imaged in live cells and can potentially be used as a rapid and quantitative cellular assay for AD drug discovery.

A folding transition underlies the emergence of membrane affinity in amyloid-ß.

Small amyloid-ß (Aß) oligomers have much higher membrane affinity compared to the monomers, but the structural origin of this functional change is not understood. We show that as monomers assemble into small n-mers (n < 10), Aß acquires a tertiary fold that is consistent with the mature fibrils. This is an early and defining transition for the aggregating peptide, and possibly underpins its altered bioactivity.

Nature of the amyloid-beta monomer and the monomer-oligomer equilibrium.

The monomer to oligomer transition initiates the aggregation and pathogenic transformation of Alzheimer amyloid-ß (Aß) peptide. However, the monomeric state of this aggregation-prone peptide has remained beyond the reach of most experimental techniques, and a quantitative understanding of this transition is yet to emerge. Here, we employ single-molecule level fluorescence tools to characterize the monomeric state and the monomer-oligomer transition at physiological concentrations in buffers mimicking the cerebrospinal fluid (CSF). Our measurements show that the monomer has a hydrodynamic radius of 0.9 ± 0.1 nm, which confirms the prediction made by some of the in silico studies. Surprisingly, at equilibrium, both Aß(40) and Aß(42) remain predominantly monomeric up to 3 µm, above which it forms large aggregates. This concentration is much higher than the estimated concentrations in the CSF of either normal or diseased brains. If Aß oligomers are present in the CSF and are the key agents in Alzheimer pathology, as is generally believed, then these must be released in the CSF as preformed entities. Although the oligomers are thermodynamically unstable, we find that a large kinetic barrier, which is mostly entropic in origin, strongly impedes their dissociation. Thermodynamic principles therefore allow the development of a pharmacological agent that can catalytically convert metastable oligomers into nontoxic monomers.

Single-photon smFRET. III. Application to pulsed illumination.

Förster resonance energy transfer (FRET) using pulsed illumination has been pivotal in leveraging lifetime information in FRET analysis. However, there remain major challenges in quantitative single-photon, single-molecule FRET (smFRET) data analysis under pulsed illumination including 1) simultaneously deducing kinetics and number of system states; 2) providing uncertainties over estimates, particularly uncertainty over the number of system states; and 3) taking into account detector noise sources such as cross talk and the instrument response function contributing to uncertainty; in addition to 4) other experimental noise sources such as background. Here, we implement the Bayesian nonparametric framework described in the first companion article that addresses all aforementioned issues in smFRET data analysis specialized for the case of pulsed illumination. Furthermore, we apply our method to both synthetic as well as experimental data acquired using Holliday junctions.

Zn(++) binding disrupts the Asp(23)-Lys(28) salt bridge without altering the hairpin-shaped cross-ß Structure of Aß(42) amyloid aggregates.

Observations like high Zn(2+) concentrations in senile plaques found in the brains of Alzheimer's patients and evidences emphasizing the role of Zn(2+) in amyloid-ß (Aß)-induced toxicity have triggered wide interest in understanding the nature of Zn(2+)-Aß interaction. In vivo and in vitro studies have shown that aggregation kinetics, toxicity, and morphology of Aß aggregates are perturbed in the presence of Zn(2+). Structural studies have revealed that Zn(2+) has a binding site in the N-terminal region of monomeric Aß, but not much is precisely known about the nature of binding of Zn(2+) with aggregated forms of Aß or its effect on the molecular structure of these aggregates. Here, we explore this aspect of the Zn(2+)-Aß interaction using one- and two-dimensional (13)C and (15)N solid-state NMR. We find that Zn(2+) causes major structural changes in the N-terminal and the loop region connecting the two ß-sheets. It breaks the salt bridge between the side chains of Asp(23) and Lys(28) by driving these residues into nonsalt-bridge-forming conformations. However, the cross-ß structure of Aß(42) aggregates remains unperturbed though the fibrillar morphology changes distinctly. We conclude that the salt bridge is not important for defining the characteristic molecular architecture of Aß(42) but is significant for determining its fibrillar morphology and toxicity.

Closing the gap on institutional delivery in northern India: a case study of how integrated machine learning approaches can enable precision public health.

INTRODUCTION: Meeting ambitious global health goals with limited resources requires a precision public health (PxPH) approach. Here we describe how integrating data collection optimisation, traditional analytics and causal artificial intelligence/machine learning (ML) can be used in a use case for increasing hospital deliveries of newborns in Uttar Pradesh, India. METHODS: Using a systematic behavioural framework we designed a large-scale survey on perceptual, interpersonal and structural drivers of women's behaviour around childbirth (n=5613). Multivariate logistic regression identified factors associated with institutional delivery (ID). Causal ML determined the cause-and-effect ordering of these factors. Variance decomposition was used to parse sources of variation in delivery location, and a supervised learning algorithm was used to distinguish population subgroups. RESULTS: Among the factors found associated with ID, the causal model showed that having a delivery plan (OR=6.1, 95% CI 6.0 to 6.3), believing the hospital is safer than home (OR=5.4, 95% CI 5.1 to 5.6) and awareness of financial incentives were direct causes of ID (OR=3.4, 95% CI 3.3 to 3.5). Distance to the hospital, borrowing delivery money and the primary decision-maker were not causal. Individual-level factors contributed 69% of variance in delivery location. The segmentation analysis showed four distinct subgroups differentiated by ID risk perception, parity and planning. CONCLUSION: These findings generate a holistic picture of the drivers and barriers to ID in Uttar Pradesh and suggest distinct intervention points for different women. This demonstrates data optimised to identify key behavioural drivers, coupled with traditional and ML analytics, can help design a PxPH approach that maximise the impact of limited resources.

Microsecond Conformational Dynamics of Biopolymers Revealed by Dynamic-Quenching Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy with Single Dye Labeling.

The single-molecule Förster resonance energy transfer (smFRET) technique is widely used for studying conformational dynamics of biopolymers. However, smFRET requires double dye labeling and is usually utilized for detecting dynamics on slow time scales (≳ milliseconds). In this Letter, we report dynamic-quenching two-dimensional fluorescence lifetime correlation spectroscopy (DQ 2D FLCS) that can elucidate the microsecond conformational dynamics of biopolymers with only single dye labeling. In DQ 2D FLCS, the difference in solvent accessibility of the labeled dye makes the fluorescence lifetime different, which is used for distinguishing different conformers. By applying DQ 2D FLCS to a singly labeled DNA hairpin, we successfully detect microsecond interconversion dynamics between the open and closed forms and evaluate the state-specific solvent accessibility of each form with Stern-Volmer analysis. Because DQ 2D FLCS is sensitive to the local structural change, it is complementary to FRET-based 2D FLCS and thus is a new, powerful tool for studying structural dynamics of biopolymers.

Fluorogenic Detection of Monoamine Neurotransmitters in Live Cells.

Monoamine neurotransmission is key to neuromodulation, but imaging monoamines in live neurons has remained a challenge. Here we show that externally added ortho-phthalaldehyde (OPA) can permeate live cells and form bright fluorogenic adducts with intracellular monoamines (e.g., serotonin, dopamine, and norepinephrine) and with L-DOPA, which can be imaged sensitively using conventional single-photon excitation in a fluorescence microscope. The peak excitation and emission wavelengths (λex = 401 nm and λem = 490 nm for serotonin; λex = 446 nm and λem = 557 nm for dopamine; and λex = 446 nm and λem = 544 nm for norepinephrine, respectively) are accessible to most modern confocal imaging instruments. The identity of monoamine containing structures (possibly neurotransmitter vesicles) in serotonergic RN46A cells is established by quasi-simultaneous imaging of serotonin using three-photon excitation microscopy. Mass spectrometry of cell extracts and of in vitro solutions helps us identify the chemical nature of the adducts and establishes the reaction mechanisms. Our method has low toxicity, high selectivity, and the ability to directly report the location and concentration of monoamines in live cells.