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The self-assembly of bacterial 30S ribosomes involves a large number of RNA folding and RNA-protein binding steps. The sequence of steps determines the overall assembly mechanism and the structure of the mechanism has ramifications for the robustness of biogenesis and resilience against kinetic traps. Thermodynamic interdependences of protein binding inferred from omission-reconstitution experiments are thought to preclude certain assembly pathways and thus enforce ordered assembly, but this concept is at odds with kinetic data suggesting a more parallel assembly landscape. A major challenge is deconvolution of the statistical distribution of intermediates that are populated during assembly at high concentrations approaching in vivo assembly conditions. To specifically resolve the intermediates formed by binding of three ribosomal proteins to the full length 16S rRNA, we introduce Fluorescence Triple-Correlation Spectroscopy (F3CS). F3CS identifies specific ternary complexes by detecting coincident fluctuations in three-color fluorescence data. Triple correlation integrals quantify concentrations and diffusion kinetics of triply labeled species, and F3CS data can be fit alongside auto-correlation and cross-correlation data to quantify the populations of 10 specific ribosome assembly intermediates. The distribution of intermediates generated by binding three ribosomal proteins to the entire native 16S rRNA included significant populations of species that were not previously thought to be thermodynamically accessible, questioning the current interpretation of the classic omission-reconstitution experiments. F3CS is a general approach for analyzing assembly and function of macromolecular complexes, especially those too large for traditional biophysical methods. [ABSTRACT FROM AUTHOR]

We have developed fluorescence triple correlation spectroscopy(F3CS) as an extension of the widely used fluorescence microscopytechnique fluorescence correlation spectroscopy. F3CS correlates threesignals at once and provides additional capabilities for the studyof systems with complex stoichiometry, kinetic processes, and irreversiblereactions. A general theory of F3CS was developed to describe theinterplay of molecular dynamics and microscope optics, leading toan analytical function to predict experimental triple correlationsof molecules that freely diffuse through the tight focus of the microscope.Experimental correlations were calculated from raw fluorescence datausing triple correlation integrals that extend multiple-tau correlationtheory to delay times in two dimensions. The quality of experimentaldata was improved by tuning specific spectroscopic parameters andemploying multiple independent detectors to minimize optoelectronicartifacts. Experiments with the reversible system of freely diffusing16S rRNA revealed that triple correlation functions contain symmetriespredicted from time-reversal arguments. Irreversible systems are shownto break these symmetries, and correlation strategies were developedto detect time-reversal asymmetries in a comprehensive way with respectto two delay times, each spanning many orders of magnitude in time.The correlation strategies, experimental approaches, and theory developedhere enable studies of the composition and dynamics of complex systemsusing F3CS. [ABSTRACT FROM AUTHOR]

Oligomerization of the HIV-1 protein Rev on the Rev Response Element (RRE) regulates nuclear export of genomic viral RNA and partially spliced viral mRNAs encoding for structural proteins. Single-molecule fluorescence spectroscopy has been used to dissect the multistep assembly pathway of this essential ribonucleo-protein, revealing dynamic intermediates and the mechanism of assembly. Assembly is initiated by binding of Rev to a high-affinity site in stem-loop IIB of the RRE and proceeds rapidly by addition of single Rev monomers, facilitated by cooperative Rev-Rev interactions on the RRE. Dwell-time analysis of fluorescence trajectories recorded during individual Rev-RRE assembly reactions has revealed the microscopic rate constants for several of the Rev monomer binding and dissociation steps. The high-affinity binding of multiple Rev monomers to the RRE is achieved on a much faster timescale than reported in previous bulk kinetic studies of Rev-RRE association, indicating that oligomerization is an early step in complex assembly. [ABSTRACT FROM AUTHOR]

Protein biosynthesis on the ribosome requires accurate reading of the genetic code in mRNA. Two conformational rearrangements in the small ribosomal subunit, a closing of the head and body around the incoming tRNA and an RNA helical switch near the mRNA decoding site, have been proposed to select for complementary base-pairing between mRNA codons and tRNA anticodons. We determined x-ray crystal structures of the WT and a hyper-accurate variant of the Escherichia coli ribosome at resolutions of 10 and 9 Å, respectively, revealing that formation of the intact 70S ribosome from its two subunits closes the conformation of the head of the small subunit independent of mRNA decoding. Moreover, no change in the conformation of the switch helix is observed in two steps of tRNA discrimination. These 70S ribosome structures indicate that mRNA decoding is coupled primarily to movement of the small subunit body, consistent with previous proposals, whereas closing of the head and the helical switch may function in other steps of protein synthesis. [ABSTRACT FROM AUTHOR]

Background Little research has been done to examine whether smokers switch to illegal or roll-your-own (RYO) cigarettes in response to a change in their relative price. Objective This paper explores how relative prices between three cigarette forms (manufactured legal, manufactured illegal and RYO cigarettes) are associated with the choice of one form over another after controlling for covariates, including sociodemographic characteristics, smokers' exposure to antismoking messaging, health warning labels and tobacco marketing. Methods Generalised estimating equations were employed to analyse the association between the price ratio of two different cigarette forms and the usage of one form over the other. Findings A 10% increase in the relative price ratio of legal to RYO cigarettes is associated with a 4.6% increase in the probability of consuming RYO cigarettes over manufactured legal cigarettes (p≤0.05). In addition, more exposure to antismoking messaging is associated with a lower odds of choosing RYO cigarettes over manufactured legal cigarettes (p≤0.05). Non-significant associations exist between the manufactured illegal to legal cigarette price ratios and choosing manufactured illegal cigarettes, suggesting that smokers do not switch to manufactured illegal cigarettes as prices of legal ones increase. However, these non-significant findings may be due to lack of variation in the price ratio measures. To improve the effectiveness of increased taxes and prices in reducing smoking, policymakers need to narrow price variability in the tobacco market. Moreover, increasing antismoking messaging reduces tax avoidance in the form of switching to cheaper RYO cigarettes in Uruguay. [ABSTRACT FROM AUTHOR]

Background Little research has been done to examine whether smokers switch to illegal or roll-your-own (RYO) cigarettes in response to a change in their relative price. Objective This paper explores how relative prices between three cigarette forms (manufactured legal, manufactured illegal and RYO cigarettes) are associated with the choice of one form over another after controlling for covariates, including sociodemographic characteristics, smokers' exposure to antismoking messaging, health warning labels and tobacco marketing. Methods Generalised estimating equations were employed to analyse the association between the price ratio of two different cigarette forms and the usage of one form over the other. Findings A 10% increase in the relative price ratio of legal to RYO cigarettes is associated with a 4.6% increase in the probability of consuming RYO cigarettes over manufactured legal cigarettes (p≤0.05). In addition, more exposure to antismoking messaging is associated with a lower odds of choosing RYO cigarettes over manufactured legal cigarettes (p≤0.05). Non-significant associations exist between the manufactured illegal to legal cigarette price ratios and choosing manufactured illegal cigarettes, suggesting that smokers do not switch to manufactured illegal cigarettes as prices of legal ones increase. However, these non-significant findings may be due to lack of variation in the price ratio measures. To improve the effectiveness of increased taxes and prices in reducing smoking, policymakers need to narrow price variability in the tobacco market. Moreover, increasing antismoking messaging reduces tax avoidance in the form of switching to cheaper RYO cigarettes in Uruguay. [ABSTRACT FROM AUTHOR]

Background: We investigated the relationship between receptivity to electronic cigarette (e-cigarette) advertisements at baseline and e-cigarette use at follow-up among adult baseline non-users of cigarettes and e-cigarettes. Methods: A nationally representative online panel was used to survey non-users of cigarettes and e-cigarettes (n = 2191) at baseline and 5-month follow-up. At baseline, respondents were shown an e-cigarette advertisement and asked if they were aware of it (exposure). Among those exposed, receptivity was self-rated for each ad using a validated scale of 1 to 5 for agreement with each of six items: "worth remembering," "grabbed my attention," "powerful," "informative," "meaningful," and "convincing." Logistic regression was used to measure the relationship between receptivity at baseline and e-cigarette use at follow-up. Results: Among baseline non-users of cigarettes and e-cigarettes, 16.6% reported exposure to e-cigarette advertisements at baseline; overall mean receptivity score was 2.77. Among baseline non-users who reported exposure to e-cigarette advertisements, incidence of e-cigarette use at follow-up was 2.7%; among baseline nonusers who reported not being exposed to e-cigarette advertisements, incidence of e-cigarette use at follow-up was 1.3%. The attributable risk percentage for e-cigarette initiation from e-cigarette advertisement exposure was 59.3%; the population attributable risk percentage from e-cigarette advertisement exposure was 22.6%. Receptivity at baseline was associated with e-cigarette use at follow-up (aOR = 1.57; 95% CI = 1.04-2.37). Conclusions: Receptivity to e-cigarette advertisements at baseline was associated with greater odds of e-cigarette use at follow-up among baseline non-users of cigarettes and e-cigarettes. Understanding the role of advertising in e-cigarette initiation could help inform public health policy. [ABSTRACT FROM AUTHOR]

Background Weight concerns are widely documented as one of the major barriers for girls and young adult women to quit smoking. Therefore, it is important to investigate whether smokers who have weight concerns respond to tobacco control policies differently than smokers who do not in terms of quit attempts, and how this difference varies by gender and country. Objective This study aims to investigate, by gender and country, whether smokers who believe that smoking helps control weight are less responsive to tobacco control policies with regards to quit attempts than those who do not. Methods We use longitudinal data from the International Tobacco Control Policy Evaluation Project in the USA, Canada, the UK and Australia to conduct the analysis. We first constructed a dichotomous indicator for smokers who have the weight control belief and then examined the disparity in policy responsiveness in terms of quit attempts by directly estimating the interaction terms of policies and the weight control belief indicator using generalised estimating equations. Findings We find that weight control belief significantly attenuates the policy impact of tobacco control measures on quit attempts among US female smokers and among UK smokers. This pattern was not found among smokers in Canada and Australia. Conclusions Although our results vary by gender and country, the findings suggest that weight concerns do alter policy responsiveness in quit attempts in certain populations. Policy makers should take this into account and alleviate weight concerns to enhance the effectiveness of existing tobacco control policies on promoting quitting smoking. [ABSTRACT FROM AUTHOR]

Background Weight concerns are widely documented as one of the major barriers for girls and young adult women to quit smoking. Therefore, it is important to investigate whether smokers who have weight concerns respond to tobacco control policies differently than smokers who do not in terms of quit attempts, and how this difference varies by gender and country. Objective This study aims to investigate, by gender and country, whether smokers who believe that smoking helps control weight are less responsive to tobacco control policies with regards to quit attempts than those who do not. Methods We use longitudinal data from the International Tobacco Control Policy Evaluation Project in the USA, Canada, the UK and Australia to conduct the analysis. We first constructed a dichotomous indicator for smokers who have the weight control belief and then examined the disparity in policy responsiveness in terms of quit attempts by directly estimating the interaction terms of policies and the weight control belief indicator using generalised estimating equations. Findings We find that weight control belief significantly attenuates the policy impact of tobacco control measures on quit attempts among US female smokers and among UK smokers. This pattern was not found among smokers in Canada and Australia. Conclusions Although our results vary by gender and country, the findings suggest that weight concerns do alter policy responsiveness in quit attempts in certain populations. Policy makers should take this into account and alleviate weight concerns to enhance the effectiveness of existing tobacco control policies on promoting quitting smoking. [ABSTRACT FROM AUTHOR]

Background: Menthol, a flavoring compound added to cigarettes, makes cigarettes more appealing to youth and inexperienced smokers and increases cigarettes' abuse liability. However, limited studies are available on menthol's role in smoking progression.Methods: To assess the association between menthol in cigarettes and progression to established smoking, we used five waves of data from the Evaluation of Public Education Campaign on Teen Tobacco Cohort Study, a nationally representative longitudinal survey of U.S. youth conducted as part of "The Real Cost" evaluation. We used discrete time survival analysis to model the occurrence of two event outcomes-progression to established, current smoking and progression to established, frequent smoking-using a logit model with a menthol use indicator as the key explanatory variable. Based on this framework, we estimated the effect of prior menthol use on the odds of smoking progression.Results: In the progression to established, current smoking model, prior menthol use was significantly associated with progression [adjusted odds ratio (aOR) = 1.80, p < .05, confidence interval (CI) = (1.03-3.16)]. While results were in a similar direction for the model of progression to established, frequent smoking, the association between prior menthol use and this progression model did not reach significance [aOR = 1.56, CI = (0.80-3.03)].Conclusion: The results suggest a relationship between using menthol cigarettes and progression from experimental to established, current smoking among youth. This study adds to a growing literature base that supports that menthol cigarettes, compared to nonmenthol cigarettes, put youth at increased risk for regular cigarette use. [ABSTRACT FROM AUTHOR]