Disentangling Subpopulations in Single-Molecule FRET and ALEX Experiments with Photon Distribution Analysis

AbstractAmong the advantages of the single-molecule approach when used to study biomolecular structural dynamics and interaction is its ability to distinguish between and independently observe minor subpopulations. In a single-molecule Förster resonance energy transfer (FRET) and alternating laser excitation diffusion experiment, the various populations are apparent in the resultant histograms. However, because histograms are calculated based on the per-burst mean FRET and stoichiometry ratio and not on the internal photon distribution, much of the acquired information is lost, thereby reducing the capabilities of the method. Here we suggest what to our knowledge is a novel statistical analysis tool that significantly enhances these capabilities, and we use it to identify and isolate static and dynamic subpopulations. Based on a kernel density estimator and a proper photon distribution analysis, for each individual burst, we calculate scores that reflect properties of interest. Specifically, we determine the FRET efficiency and brightness ratio distributions and use them to reveal 1), the underlying structure of a two-state DNA-hairpin and a DNA hairpin that is bound to DNA origami; 2), a minor doubly labeled dsDNA subpopulation concealed in a larger singly labeled dsDNA; and 3), functioning DNA origami motors concealed within a larger subpopulation of defective motors. Altogether, these findings demonstrate the usefulness of the proposed approach. The method was developed and tested using simulations, its rationality is described, and a computer algorithm is provided

Parental psychopathology and the risk of suicidal behavior in their offspring: results from the World Mental Health surveys

Prior research suggests that parental psychopathology predicts suicidal behavior among offspring; however, the more fine-grained associations between specific parental disorders and distinct stages of the pathway to suicide are not well-understood. We set out to test the hypothesis that parental disorders associated with negative mood would predict offspring suicide ideation, whereas disorders characterized by impulsive-aggression (e,g., antisocial personality) and anxiety/agitation (e.g., panic disorder) would predict which offspring act on their suicide ideation and make a suicide attempt. Data were collected during face-to-face interviews conducted on nationally representative samples (N=55,299; age 18+) from 21 countries around the world. We tested the associations between a range of parental disorders and the onset and persistence over time (i.e., time-since-most-recent-episode controlling for age-of-onset and time-since-onset) of subsequent suicidal behavior (suicide ideation, plans, and attempts) among offspring. Analyses tested bivariate and multivariate associations between each parental disorder and distinct forms of suicidal behavior. Results revealed that each parental disorder examined increased the risk of suicide ideation among offspring, parental generalized anxiety and depression emerged as the only predictors of the onset and persistence (respectively) of suicide plans among offspring with ideation, whereas parental anti-social personality and anxiety disorders emerged as the only predictors of the onset and persistence of suicide attempts among ideators. A dose-response relation between parental disorders and respondent risk of suicide ideation and attempt also was found. Parental death by suicide was a particularly strong predictor of persistence of suicide attempts among offspring. These associations remained significant after controlling for comorbidity of parental disorders and for the presence of mental disorders among offspring. These findings should inform future explorations of the mechanisms of inter-generational transmission of suicidal behavior.Psycholog

Conformational Dynamics of DNA Hairpins at Millisecond Resolution Obtained from Analysis of Single-Molecule FRET Histograms

Here we provide high resolution study of DNA hairpin dynamics achieved by probability distribution analysis (PDA) of diffusion-based single-molecule Förster resonance energy transfer (sm-FRET) histograms. The opening and closing rates of three hairpins both free and attached to DNA origami were determined. The agreement with rates previously obtained using the total internal reflection (TIRF) technique and between free hairpins and hairpins attached to origami validated the PDA and demonstrated that the origami had no influence on the hairpin dynamics. From comparison of rates of four DNA hairpins, differing only in stem sequence, and from comparison with rates calculated using nearest-neighbor method and standard transition state theory, we conclude that the unfolding reaction resembles that of melting of DNA duplex with a corresponding sequence and that the folding reaction depends on counterion concentration and not on stem sequence. Our validation and demonstration of the PDA method will encourage its implementation in future high-resolution dynamic studies of freely diffusing biomolecules

Rational Design of DNA Motors: Fuel Optimization through Single-Molecule Fluorescence

While numerous DNA-based molecular machines have been developed in recent years, high operational yield and speed remain a major challenge. To understand the reasons for the limited performance, and to find rational solutions, we applied single-molecule fluorescence techniques and conducted a detailed study of the reactions involved in the operation of a model system comprised of a bipedal DNA walker that strides on a DNA origami track powered by interactions with fuel and antifuel strands. Analysis of the kinetic profiles of the leg-lifting reactions indicates a pseudo-first-order antifuel binding mechanism leading to a rapid and complete leg-lifting, indicating that the fuel-removal reaction is not responsible for the 1% operational yield observed after six steps. Analysis of the leg-placing reactions showed that although increased concentrations of fuel increase the reaction rate, they decrease the yield by consecutively binding the motor and leading to an undesirable trapped state. Recognizing this, we designed asymmetrical hairpin-fuels that by regulating the reaction hierarchy avoid consecutive binding. Motors operating with the improved fuels show 74% yield after 12 consecutive reactions, a dramatic increase over the 1% observed for motors operating with nonhairpin fuels. This work demonstrates that studying the mechanisms of the reactions involved in the operation of DNA-based molecular machines using single-molecule fluorescence can facilitate rationally designed improvements that increase yield and speed and promote the applicability of DNA-based machines

Studying the Structural Dynamics of Bipedal DNA Motors with Single-Molecule Fluorescence Spectroscopy

We present a test case example of a detailed single-molecule fluorescence study of one of the most sophisticated and complex DNA devices introduced to date, a recently published autonomous bipedal DNA motor. We used the diffusion-based single-molecule Förster resonance energy transfer technique, coupled to alternating laser excitation (sm-FRET–ALEX), to monitor the motor assembly and operation. The study included verification of the formation of the correct structures, and of the correct motor operation, determination of the formation and stepping reaction yields, and identification of side products. Finally, the mechanisms of the motor assembly and operation were elucidated by measuring the reaction kinetics profile of track-walker binding and of lifting of the walker’s leg upon fuel addition. The profiles revealed a fast phase, in which about half of the reaction was completed, followed by a slow phase which adds somewhat to the yield, reflecting the incomplete motor assembly and operation identified in the equilibrium experiments. Although further study is needed to fully understand the reasons for the incomplete assembly and operation, this work demonstrates that single-molecule fluorescence, based on its ability to provide detailed <i>in situ</i> structural dynamics information, inaccessible for traditional methods, constitutes an excellent tool for chaperoning the development of DNA-based technology

The effectiveness of supported employment for people with severe mental illness: a randomised controlled trial

BACKGROUND: The value of the individual placement and support (IPS) programme in helping people with severe mental illness gain open employment is unknown in Europe. Our aim was to assess the effectiveness of IPS, and to examine whether its effect is modified by local labour markets and welfare systems. METHODS: 312 patients with severe mental illness were randomly assigned in six European centres to receive IPS (n=156) or vocational services (n=156). Patients were followed up for 18 months. The primary outcome was the difference between the proportions of people entering competitive employment in the two groups. The heterogeneity of IPS effectiveness was explored with prospective meta-analyses to establish the effect of local welfare systems and labour markets. Analysis was by intention to treat. This study is registered with ClinicalTrials.gov, with the number NCT00461318. FINDINGS: IPS was more effective than vocational services for every vocational outcome, with 85 (55%) patients assigned to IPS working for at least 1 day compared with 43 (28%) patients assigned to vocational services (difference 26.9%, 95% CI 16.4-37.4). Patients assigned to vocational services were significantly more likely to drop out of the service and to be readmitted to hospital than were those assigned to IPS (drop-out 70 [45%] vs 20 [13%]; difference -32.1% [95% CI -41.5 to -22.7]; readmission 42 [31%] vs 28 [20%]; difference -11.2% [-21.5 to -0.90]). Local unemployment rates accounted for a substantial amount of the heterogeneity in IPS effectiveness. INTERPRETATION: Our demonstration of the effectiveness of IPS in widely differing labour market and welfare contexts confirms this service to be an effective approach for vocational rehabilitation in mental health that deserves investment and further investigation

Photon-by-Photon Hidden Markov Model Analysis for Microsecond Single-Molecule FRET Kinetics

The function of biological macromolecules involves large-scale conformational dynamics spanning multiple time scales, from microseconds to seconds. Such conformational motions, which may involve whole domains or subunits of a protein, play a key role in allosteric regulation. There is an urgent need for experimental methods to probe the fastest of these motions. Single-molecule fluorescence experiments can in principle be used for observing such dynamics, but there is a lack of analysis methods that can extract the maximum amount of information from the data, down to the microsecond time scale. To address this issue, we introduce H²MM, a maximum likelihood estimation algorithm for photon-by-photon analysis of single-molecule fluorescence resonance energy transfer (FRET) experiments. H²MM is based on analytical estimators for model parameters, derived using the Baum–Welch algorithm. An efficient and effective method for the calculation of these estimators is introduced. H²MM is shown to accurately retrieve the reaction times from ∼1 s to ∼10 μs and even faster when applied to simulations of freely diffusing molecules. We further apply this algorithm to single-molecule FRET data collected from Holliday junction molecules and show that at low magnesium concentrations their kinetics are as fast as ∼10⁴ s^–1. The new algorithm is particularly suitable for experiments on freely diffusing individual molecules and is readily incorporated into existing analysis packages. It paves the way for the broad application of single-molecule fluorescence to study ultrafast functional dynamics of biomolecules

Predictors of employment for people with severe mental illness: results of an international six-centre RCT

BACKGROUND: An international six-centre randomised controlled trial comparing individual placement and support (IPS) with usual vocational rehabilitation for people with serious mental illness found IPS to be more effective for all vocational outcomes. AIMS: To determine which patients with severe mental illness do well in vocational services and which process and service factors are associated with better outcomes. METHOD: Patient characteristics and early process variables were tested as predictors of employment outcomes. Service characteristics were explored as predictors of the effectiveness of IPS. RESULTS: Patients with previous work history, fewer met social needs and better relationships with their vocational workers were more likely to obtain employment and work for longer. Remission and swifter service uptake were associated with working more. Having an IPS service closer to the original IPS model was the only service characteristic associated with greater effectiveness. CONCLUSIONS: The IPS service was found to be more effective for all vocational outcomes. In addition, maintaining high IPS fidelity and targeting relational skills would be a valuable focus for all vocational interventions, leading to improved employment outcomes. Motivation to find work may be decreased by satisfaction with current life circumstances