Changes in Retail Tobacco Product Availability Following a Chicago City Ordinance Restricting Sales of Menthol and Other Flavored Tobacco Products Near Schools.

BACKGROUND: In April 2016, the City of Chicago implemented an ordinance restricting the sale of all flavored (including menthol) tobacco products (FTPs), including electronic cigarettes, at retailers located within 500 feet of any public, private, or alternative elementary, middle ("primary"), or high ("secondary") school. We examined changes in retail availability of FTPs from before to after policy implementation among policy-affected retailers compared with retailers not subject to the policy. METHOD: Observational data were collected in June to September 2015 (Wave 1; pre-policy) and November to December 2016 (Wave 2; post-policy) from a panel of 194 randomly selected policy-area stores (located within 500 feet of a school), and a panel of 199 randomly selected comparison-area stores (located more than 500 feet from a school). Using generalized estimation equation regression, we assessed differences in FTP availability changes across study areas. RESULTS: We observed a statistically significant policy effect on FTP availability (Area × Wave interaction, p < .05); however, more than half of policy-area retailers continued to display at least one FTP after policy implementation (87.11% at Wave 1, 57.73% at Wave 2, p < .05). Similar reductions were seen for the availability of flavored cigarillos/little cigars and menthol cigarettes, while policy effects varied across store types. DISCUSSION: FTP availability reductions appear to be associated with policy implementation, but FTPs remained readily available at retailers subject to the policy. This study contributes to the evidence base indicating that policies with exclusions or exemptions for certain flavors, products, store types, or retailer locations have a limited effect on retail availability of FTPs.

Examining the role of menthol cigarettes in progression to established smoking among youth.

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.

Association Between Media Doses of the Tips From Former Smokers Campaign and Cessation Behaviors and Intentions to Quit Among Cigarette Smokers, 2012-2015.

BACKGROUND: Since 2012, the Centers for Disease Control and Prevention (CDC) has implemented Tips From Former Smokers ( Tips), the first federally funded tobacco education campaign in the United States. To date, there are no evaluations of its long-term impact. AIMS: To assess the impact of varied doses of the Tips campaign from 2012 through 2015 on cessation-related behaviors and intentions among U.S. smokers. METHOD: We used a national probability-based online survey of cigarette smokers ( n = 22,189) and recent quitters ( n = 776) to examine associations between doses of Tips advertising, measured by gross rating points (GRPs), and intentions to quit smoking in the next 30 days and quit attempts within the past 3 months. A curvilinear (i.e., square root) functional form of GRPs was used to capture patterns of diminishing effects at higher GRP levels. RESULTS: An increase of 1,000 quarterly Tips GRPs at the media market level was associated with increased odds of making a quit attempt in the past 3 months (adjusted odds ratio = 1.23, p < .001) and increased odds of intending to quit in the next 30 days (adjusted odds ratio = 1.17, p = .030). DISCUSSION: Results suggest that CDC-recommended media buys of 800 to 1,000 GRPs per quarter are sufficient to generate statistically significant increases in the likelihood of quit attempts in the past quarter. CONCLUSIONS: The Tips campaign has had a substantial impact on cessation behaviors among U.S. adult smokers over time. These data support the continued use of graphic and/or emotional media campaigns that encourage smokers to quit to further reduce tobacco use in the United States.

A longitudinal study of the relationship between receptivity to e-cigarette advertisements and e-cigarette use among baseline non-users of cigarettes and e-cigarettes, United States.

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 non-users 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.

Evaluation of the National Tips From Former Smokers Campaign: the 2014 Longitudinal Cohort.

INTRODUCTION: Since 2012, the Centers for Disease Control and Prevention has aired a national tobacco education campaign to encourage quitting, Tips From Former Smokers (Tips), which consists of graphic antismoking advertisements that feature former cigarette smokers. We evaluated phase 2 of the 2014 campaign by using a nationally representative longitudinal cohort. METHODS: Cigarette smokers who participated in a baseline survey were re-contacted for follow-up (n = 4,248) approximately 4 months later, immediately after the campaign's conclusion. The primary outcomes were incidence of a quit attempt in the previous 3 months, intention to quit within 30 days, and intention to quit within 6 months during the postcampaign period. We used multivariate logistic regression models to estimate the odds of each outcome. We also stratified models by race/ethnicity, education, and mental health status. Postcampaign rates of quit attempts, intentions to quit, and sustained quits were also estimated. RESULTS: Exposure to the campaign was associated with increased odds of a quit attempt in the previous 3 months (OR, 1.17; P = .03) among baseline smokers and intentions to quit within the next 6 months (OR, 1.28; P = .01) among current smokers at follow-up. The Tips campaign was associated with an estimated 1.83 million additional quit attempts, 1.73 million additional smokers intending to quit within 6 months, and 104,000 sustained quits of at least 6 months. CONCLUSION: The Tips campaign continued to have a significant impact on cessation-related behaviors, providing further justification for the continued use of tobacco education campaigns to accelerate progress toward the goal of reducing adult smoking in the United States.

The use of legal, illegal and roll-your-own cigarettes to increasing tobacco excise taxes and comprehensive tobacco control policies: findings from the ITC Uruguay Survey.

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.

Weight control belief and its impact on the effectiveness of tobacco control policies on quit attempts: findings from the ITC 4 Country Survey.

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.

Vectorized data acquisition and fast triple-correlation integrals for Fluorescence Triple Correlation Spectroscopy.

Fluorescence Correlation Spectroscopy (FCS) is widely used to quantitate reaction rates and concentrations of molecules in vitro and in vivo. We recently reported Fluorescence Triple Correlation Spectroscopy (F3CS), which correlates three signals together instead of two. F3CS can analyze the stoichiometries of complex mixtures and detect irreversible processes by identifying time-reversal asymmetries. Here we report the computational developments that were required for the realization of F3CS and present the results as the Triple Correlation Toolbox suite of programs. Triple Correlation Toolbox is a complete data analysis pipeline capable of acquiring, correlating and fitting large data sets. Each segment of the pipeline handles error estimates for accurate error-weighted global fitting. Data acquisition was accelerated with a combination of off-the-shelf counter-timer chips and vectorized operations on 128-bit registers. This allows desktop computers with inexpensive data acquisition cards to acquire hours of multiple-channel data with sub-microsecond time resolution. Off-line correlation integrals were implemented as a two delay time multiple-tau scheme that scales efficiently with multiple processors and provides an unprecedented view of linked dynamics. Global fitting routines are provided to fit FCS and F3CS data to models containing up to ten species. Triple Correlation Toolbox is a complete package that enables F3CS to be performed on existing microscopes.

Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy.

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 interdependencies 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.

The spectroscopic basis of fluorescence triple correlation spectroscopy.

We have developed fluorescence triple correlation spectroscopy (F3CS) as an extension of the widely used fluorescence microscopy technique fluorescence correlation spectroscopy. F3CS correlates three signals at once and provides additional capabilities for the study of systems with complex stoichiometry, kinetic processes, and irreversible reactions. A general theory of F3CS was developed to describe the interplay of molecular dynamics and microscope optics, leading to an analytical function to predict experimental triple correlations of molecules that freely diffuse through the tight focus of the microscope. Experimental correlations were calculated from raw fluorescence data using triple correlation integrals that extend multiple-tau correlation theory to delay times in two dimensions. The quality of experimental data was improved by tuning specific spectroscopic parameters and employing multiple independent detectors to minimize optoelectronic artifacts. Experiments with the reversible system of freely diffusing 16S rRNA revealed that triple correlation functions contain symmetries predicted from time-reversal arguments. Irreversible systems are shown to break these symmetries, and correlation strategies were developed to detect time-reversal asymmetries in a comprehensive way with respect to two delay times, each spanning many orders of magnitude in time. The correlation strategies, experimental approaches, and theory developed here enable studies of the composition and dynamics of complex systems using F3CS.

RNA-protein binding kinetics in an automated microfluidic reactor.

Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA-protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic 'Riboreactor' has been designed and constructed to facilitate the study of kinetics of RNA-protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA-protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome.

HIV-1 Rev protein assembles on viral RNA one molecule at a time.

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 ribonucleoprotein, 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.

X-ray crystal structures of the WT and a hyper-accurate ribosome from Escherichia coli.

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 A, 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.