Selective Reduction of Socioeconomic Disparities in the Experimental Tobacco Marketplace: Effects of Cigarette and E-cigarette Flavor Restrictions.

INTRODUCTION: Cigarette smoking accounts for >30% of the socioeconomic gap in life expectancy. Flavored restrictions claim to promote equity; however, no previous studies have compared the effect of cigarette and e-cigarette flavor restrictions among individuals who smoke with lower and higher socioeconomic status (SES). AIMS AND METHODS: In a between-group within-subject design, individuals with lower (n = 155) and higher (n = 125) SES completed hypothetical purchasing trials in the experimental tobacco marketplace (ETM). Conditions were presented in a 2 × 2 factorial design (cigarette flavors restricted or unrestricted and e-cigarette flavors restricted or unrestricted) with increasing cigarette prices across trials. RESULTS: Results show (1) SES differences in cigarette, e-cigarette, and NRT purchases under unrestricted policies, with lower SES showing higher cigarette demand and lower e-cigarette and NRT substitution than higher SES, (2) cigarette restrictions decreased cigarette and increased NRT purchases among lower SES, but no significant changes among higher SES, (3) decreased SES differences in cigarette demand under cigarette restrictions, but persistence under e-cigarette restrictions or their combination, (4) persistence of SES differences in e-cigarette purchases when all restrictions were enforced, and (5) waning of SES differences in NRT purchasing under all restrictions. CONCLUSIONS: Flavor restrictions differentially affected individuals based on SES. Within-group comparisons demonstrated restrictions significantly impacted lower SES, but not higher SES. Between-group comparisons showed SES differences in cigarette purchasing decreased under cigarette restrictions, but persisted under e-cigarette-restrictions or their combination. Additionally, SES differences in NRT substitution decreased under flavor restrictions. These findings highlight the utility of the ETM to investigate SES disparities. IMPLICATIONS: With increasing trends of socioeconomic differences in smoking prevalence and cessation rates, smoking-related health disparities are expected to continue to widen. Restricting menthol flavor in cigarettes while enhancing the availability and affordability of NRT have the potential to alleviate SES disparities in tobacco use, therefore, positively impacting health equity. However, this effect may depend on flavor availability in other tobacco products.

Illegal Experimental Tobacco Marketplace II: effects of vaping product bans - findings from the 2020 International Tobacco Control Project.

SIGNIFICANCE: Restrictive e-cigarette policies may increase purchases from illegal sources. The Illegal Experimental Tobacco Marketplace (IETM) allows examination of how restrictions impact illegal purchases. We investigated (1) the effect of a vaping ban, total flavour vaping ban and partial flavour vaping ban on the probability of purchasing illegal vaping products among different regulatory environments (USA, Canada and England) and tobacco user types (cigarette smokers, dual users and e-cigarette users); and (2) the relation between ban endorsement and illegal purchases. METHODS: Participants (N=459) from the International Tobacco Control Survey rated their support of bans and chose to purchase from a hypothetical legal experimental tobacco marketplace or IETM under control and the three ban conditions. RESULTS: In total, 25% of cigarette smokers, 67% of dual users and 79% of e-cigarette users made IETM purchases. Cross-country comparisons depicted dual users from Canada (OR: 19.8), and e-cigarette users from the USA (OR: 12.9) exhibited higher illegal purchases odds than the same user type in England. Within-country comparisons showed e-cigarette and dual users are more likely to purchase from the IETM than cigarette smokers in the most restrictive condition, with the largest effects in e-cigarette users (England-OR: 1722.6, USA-OR: 22725.3, Canada-OR: 6125.0). Increased opposition towards partial or total flavour ban was associated with increased IETM purchasing in the corresponding condition. CONCLUSIONS: Vaping restrictions may shift users' preference to the illegal marketplace in a regulatory environment. Evidence of the IETM generalisability in a geographically dispersed sample enhances its utility in tobacco regulatory science.

Lessons learned in the Apple Heart Study and implications for the data management of future digital clinical trials.

The digital clinical trial is fast emerging as a pragmatic trial that can improve a trial's design including recruitment and retention, data collection and analytics. To that end, digital platforms such as electronic health records or wearable technologies that enable passive data collection can be leveraged, alleviating burden from the participant and study coordinator. However, there are challenges. For example, many of these data sources not originally intended for research may be noisier than traditionally obtained measures. Further, the secure flow of passively collected data and their integration for analysis is non-trivial. The Apple Heart Study was a prospective, single-arm, site-less digital trial designed to evaluate the ability of an app to detect atrial fibrillation. The study was designed with pragmatic features, such as an app for enrollment, a wearable device (the Apple Watch) for data collection, and electronic surveys for participant-reported outcomes that enabled a high volume of patient enrollment and accompanying data. These elements led to challenges including identifying the number of unique participants, maintaining participant-level linkage of multiple complex data streams, and participant adherence and engagement. Novel solutions were derived that inform future designs with an emphasis on data management. We build upon the excellent framework of the Clinical Trials Transformation Initiative to provide a comprehensive set of guidelines for data management of the digital clinical trial that include an increased role of collaborative data scientists in the design and conduct of the modern digital trial.

The Illegal Experimental Tobacco Marketplace I: Effects of Vaping Product Bans.

OBJECTIVES: Banning vaping products may have unintended outcomes, such as increased demand for illegal products. This study experimentally examined the effects of a vaping ban and a flavored vaping ban on the probability of purchasing illicit vaping products, and factors affecting purchasing from a hypothetical illegal marketplace. METHODS: A crowdsourced sample of exclusive cigarette smokers, exclusive e-cigarette users, and frequent dual users (n = 150) completed hypothetical purchasing trials in an Experimental Tobacco Marketplace under three conditions (no ban, vaping ban, and flavored vaping ban). Participants chose to purchase in a hypothetical legal experimental tobacco marketplace (LETM) or illegal experimental tobacco marketplace (IETM). Vaping products were available in each marketplace depending on the condition. Other tobacco products were always available in the LETM. A hypothetical illicit purchase task with five fine amounts assessed the effect of monetary penalties. RESULTS: Participants from all groups were more likely to purchase from the IETM when product availability in the LETM was more restricted, with e-cigarette users being most affected. The likelihood of purchasing illegal products was systematically decreased as monetary penalties associated with the IETM increased, with e-cigarette users showing greater persistence in defending their illicit purchases. CONCLUSIONS: Restricting vaping products from the marketplace may shift preference towards purchasing vaping products in the illegal marketplace. Nevertheless, penalties imposed on consumer's behavior might be effective in preventing illicit trade. The IETM is a methodological extension that supports the utility and flexibility of the ETM as a framework for understanding the impact of different tobacco regulatory policies. IMPLICATIONS: This study suggests that limiting or banning vaping products as a possible strategy to reduce the adverse effects of vaping products could result in some tobacco-users seeking banned products from illegal sources. Monetary fines were shown to reduce illegal purchases. Therefore, policymakers should consider implementing strategies that may mitigate illegal purchases.

Bleak present, bright future: II. Combined effects of episodic future thinking and scarcity on delay discounting in adults at risk for type 2 diabetes.

The present study sought to determine if episodic future thinking (EFT) can decrease delay discounting (DD) and demand for fast food under simulations of economic scarcity in adults at risk for diabetes (i.e., overweight/obese and with hemoglobin A1c values in, or approaching, the prediabetic range). Across two sessions, participants completed assessments of DD and food demand at baseline and while prompted to: (1) engage in either EFT or control episodic recent thinking, and (2) while reading a brief narrative describing either economic scarcity or neutral income conditions. Results showed that EFT significantly reduced DD, whereas the economic scarcity narrative significantly increased DD; no significant interaction between EFT and scarcity was observed. No significant effect of either EFT or scarcity was observed on food demand. We conclude that EFT decreases DD even when challenged by simulated economic scarcity in adults at risk for diabetes. The absence of a significant interaction between EFT and scarcity suggests that these variables operate independently to influence DD in opposing directions. Effects of EFT and economic scarcity on food demand require further study. The present study was registered on clinicaltrials.gov (NCT03664726).

Delay Discounting, Glycemic Regulation and Health Behaviors in Adults with Prediabetes.

The majority of people with prediabetes transition to type 2 diabetes. Research has suggested that persons with type 2 diabetes are likely to discount the future and focus on immediate rewards. This study was designed to assess whether this process of delay discounting (DD) is associated with glycemic regulation, medication adherence and eating and exercise behaviors in adults with prediabetes. Participants included 81 adults with prediabetes who were also prescribed hypertension or dyslipidemia drugs, which is common for people with prediabetes. Participants completed adjusting amount DD $100 and $1000 tasks, as well assessments of glycemic control (Hemoglobin (Hb) A1c), medication adherence, diet quality, and objectively measured physical activity. Relationships between DD and these variables were assessed. Results showed higher rates of DD were related to higher HbA1c; as well as poorer medication adherence, lower diet quality and lower physical activity. Hierarchical regression showed that the association between minority status, a known risk factor for type 2 diabetes, was moderated by DD, as minorities with higher DD had greater HbA1c values. Delay discounting may represent a novel target to prevent progression from prediabetes to type 2 diabetes.

Does Episodic Future Thinking Repair Immediacy Bias at Home and in the Laboratory in Patients With Prediabetes?

OBJECTIVE: This study aimed to determine if episodic future thinking (EFT) can decrease delay discounting (DD) among adults with prediabetes both in and out of the laboratory. DD measures how much the value of a reinforcer decreases as a function of the delay to receive it. METHODS: Adults with prediabetes (n = 67) completed a three-session study. At session 1, baseline measures (including DD) were collected. At sessions 2 and 3, participants were prompted to engage in either EFT or control episodic thinking (CET) while completing DD and other measures. In addition, between the completion of sessions 2 and 3, participants engaged in EFT or CET at home and completed DD tasks remotely via smartphones or other Internet-connected devices. RESULTS: Results showed significant -1.2759 (-20.24%) reductions in DD in the EFT group compared with a + 0.0287 (+0.46%) DD increase in the CET group (p = .0149) in the laboratory; and -0.4095 (-8.85%) reduction in DD in the EFT group compared with a + 0.2619 (+5.64%) increase in the CET group (p = .011) at home. Working memory (measured by Backwards Corsi and Digit Span) was found to moderate the effects of EFT on some measures of DD. EFT did not change measures from the food purchase task or a food ad libitum procedure. CONCLUSIONS: Results show that EFT decreases DD in and out of the laboratory and supports the further exploration of EFT as an intervention for prediabetes and related chronic diseases. CLINICAL TRIAL REGISTRATION: NCT03664726.

The Experimental Tobacco Marketplace: Demand and Substitutability as a Function of Cigarette Taxes and e-Liquid Subsidies.

INTRODUCTION: The experimental tobacco marketplace (ETM) approximates real-world situations by estimating the effects of several, concurrently available products and policies on budgeted purchasing. Although the effects of increasing cigarette price on potentially less harmful substitutability are well documented, the effects of other, nuanced pricing policies remain speculative. This study used the ETM as a tool to assess the effects of two pricing policies, conventional cigarette taxation and e-liquid subsidization, on demand and substitutability. METHODS: During sampling periods, participants were provided 2-day samples of 24 mg/mL e-liquid, after which ETM purchase sessions occurred. Across two ETM sessions, conventional cigarettes were taxed or e-liquid was subsidized in combination with increasing cigarette price. The other four available products were always price constant and not taxed or subsidized. RESULTS: E-liquid functioned as a substitute for conventional cigarettes across all conditions. Increasing cigarette taxation and e-liquid subsidization increased the number of participants for which e-liquid functioned as a substitute. Cigarette taxation decreased cigarette demand, by decreasing demand intensity, and marginally increased the initial intensity of e-liquid substitution, but did not affect the functions' slopes (substitutability). E-liquid subsidization resulted in large increases in the initial intensity of e-liquid substitution, but did not affect e-liquid substitutability nor cigarette demand. IMPLICATIONS: 24 mg/mL e-cigarette e-liquid was the only product to significantly substitute for cigarettes in at least one condition throughout the experiment; it functioned as a significant substitute throughout all four tax and all four subsidy conditions. Increasing cigarette taxes decreased cigarette demand through decreases in demand intensity but did not affect e-cigarette substitution. Increasing e-liquid subsidies increased e-liquid initial intensity of substitution but did not affect cigarette demand. CONCLUSIONS: This study extended research on the behavioral economics of conventional cigarette demand and e-liquid substitutability in a complex marketplace. The results suggest that the most efficacious method to decrease conventional cigarette purchasing and increase e-liquid purchasing may involve greatly increasing cigarette taxes while also increasing the value of e-liquid through potentially less harmful product subsidization or differential taxation.

Reinforcer pathology: Common neural substrates for delay discounting and snack purchasing in prediabetics.

Reinforcer pathology theory stipulates that individuals with both (a) high preference for smaller, immediate over larger, delayed rewards; and (b) high demand for unhealthy commodities are uniquely susceptible to poor health outcomes. Specifically, two behavioral economic tasks (delay discounting, assessing preference for smaller, immediate or larger, delayed rewards; and purchasing, assessing purchases of commodities over changes in price) have been independently associated with conditions such as overweight/obesity and problem substance use. In the present study, we examined possible shared neural regions involved in the processes of delay discounting and demand for snack foods in a prediabetic sample. Fifty-four participants completed both of these tasks. Conjunction between delay discounting and purchasing task results indicates substantial common neural substrates recruited during these two tasks, consistent with interpretations of executive control, interoception, and attention, in the prefrontal cortex, insula, and frontoparietal cortex (superior/middle frontal cortex and superior/inferior parietal lobules), respectively. Collectively, these results suggest possible neural substrates in which the two behavioral risk factors of reinforcer pathology may interact during real-world decision-making in prediabetes.

Experimental tobacco marketplace: substitutability of e-cigarette liquid for cigarettes as a function of nicotine strength.

BACKGROUND: The experimental tobacco marketplace (ETM) provides a method to estimate, prior to implementation, the effects of new products or policies on purchasing across various products in a complex tobacco marketplace. We used the ETM to examine the relationship between nicotine strength and substitutability of alternative products for cigarettes to contribute to the literature on regulation of e-liquid nicotine strength. METHODS: The present study contained four sampling and four ETM purchasing sessions. During sampling sessions, participants were provided 1 of 4 e-liquid strengths (randomised) to sample for 2 days followed by an ETM purchasing session. The nicotine strength sampled in the 2 days prior to an ETM session was the same strength available for purchase in the next ETM. Each participant sampled and could purchase 0 mg/mL, 6 mg/mL, 12 mg/mL and 24 mg/mL e-liquid, among other products, during the study. RESULTS: Cigarette demand was unaltered across e-liquid strength. E-liquid was the only product to substitute for cigarettes across more than one e-liquid strength. Substitutability increased as a function of e-liquid strength, with the 24 mg/mL displaying the greatest substitutability of all products. CONCLUSIONS: The present study found that e-liquid substitutability increased with nicotine strength, at least up to 24 mg/mL e-liquid. However, the effects of e-liquid nicotine strength on cigarette purchasing were marginal and total nicotine purchased increased as e-liquid nicotine strength increased.

Electronic cigarette substitution in the experimental tobacco marketplace: A review.

The evolution of science derives, in part, from the development and use of new methods and techniques. Here, we discuss one development that may have impact on the understanding of tobacco regulatory science: namely, the application of behavioral economics to the complex tobacco marketplace. The purpose of this paper is to review studies that examine conditions impacting the degree to which electronic nicotine delivery system (ENDS) products substitute for conventional cigarettes in the Experimental Tobacco Marketplace (ETM). Collectively, the following factors constitute the current experimental understanding of conditions that will affect ENDS use and substitution for conventional cigarettes: increasing the base price of conventional cigarettes, increasing taxation of conventional cigarettes, subsidizing the price of ENDS products, increasing ENDS nicotine strength, and providing narratives that illustrate the potential health benefits of ENDS consumption in lieu of conventional cigarettes. Each of these factors are likely moderated by consumer characteristics, which include prior ENDS use, ENDS use risk perception, and gender. Overall, the ETM provides a unique method to explore and identify the conditions by which various nicotine products may interact with one another that mimics the real world. In addition, the ETM permits the efficacy of a broad range of potential nicotine policies and regulations to be measured prior to governmental implementation.

Effects of Filter Ventilation on Behavioral Economic Demand for Cigarettes: A Preliminary Investigation.

Introduction: The majority of cigarettes sold in the United States and abroad feature filter ventilation holes designed to dilute mainstream smoke. Although initially intended to produce a safer cigarette, data instead suggest that filter ventilation increases total harm from smoking. In the present study, we examined the effects of blocking ventilation holes on behavioral economic demand for cigarettes (i.e., consumption as a function of price). Methods: In a within-subjects design, regular smokers (N = 15) of ventilated cigarettes sampled vent-blocked cigarettes for 3 days. Subsequently, they completed three sessions in which they used an experimental income to purchase vent-blocked and/or control cigarettes across a range of prices. Participants also completed the Drug Effects/Liking Scale. Results: In sessions in which only one cigarette type was available, demand measures were undifferentiated between cigarette types. However, in sessions in which both cigarettes were available at equivalent prices, significantly greater preference for ventilated control cigarettes emerged in demand measures. Regardless of session type, participants also rated vent-blocked cigarettes more poorly in the Drug Effects/Liking Scale (more bad effects, fewer good effects, and less liking, desire, and less likely to use again). Conclusions: Removing filter ventilation reduced cigarette abuse liability, as measured by behavioral economic demand and the Drug Effects/Liking Scale. However, reduced demand was only apparent when both cigarette types were concurrently available. This selective effect suggests that regulatory action banning filter ventilation would only reduce cigarette consumption when effective substitutes for vent-blocked cigarettes are available. Implications: This preliminary study indicates that regulatory action designed to ban or restrict cigarette filter ventilation may decrease cigarette abuse liability as measured by both behavioral economic demand and self-report measures. However, effects of removing filter ventilation on demand measures appear to depend on concurrent availability of alternative, preferred cigarette types.

Episodic future thinking reduces delay discounting and cigarette demand: an investigation of the good-subject effect.

Episodic future thinking (EFT), an intervention involving mental simulation of future events, has been shown to reduce both delay discounting and cigarette self-administration. In the present study, we extended these findings by showing that EFT in a web-based sample of smokers reduces delay discounting and intensity of demand for cigarettes (ad libitum consumption) in a hypothetical purchase task. No effect was observed on elasticity of demand (sensitivity to price) or cigarette craving. We also explored whether demand characteristics (specifically, the "good-subject" effect) might be responsible for observed effects. EFT participants were significantly better able than control participants to discern the experimental hypothesis. However, EFT participants were not better than controls at identifying whether they had been assigned to the experimental group and, likewise, showed no differences in attitudes about the experiment and experimenter. Importantly, effects of EFT on delay discounting and demand remained significant even when controlling for measures of demand characteristics, indicating that EFT's effects are independent of participants' perceptions about the experiment.

Binary components of food reinforcement: Amplitude and persistence.

BACKGROUND: Demand curves provide an index of how reinforcing a food is. Research examining the latent structure of alcohol and tobacco reinforcement identified two underlying components of reinforcement, amplitude and persistence. No research has assessed latent structure of food reinforcement and how these factors are related to BMI. SUBJECTS AND METHODS: Participants were 297 adults from two studies that completed food purchasing tasks to assess the following measures of relative reinforcing efficacy (RRE) of food: intensity (Q0): purchases made when the food was free or of very minimal price, Omax: maximum expenditure (purchases*price), Pmax: price point where maximum expenditure was observed, breakpoint: first price where 0 purchases are made, and demand elasticity (α): quantitative non-linear relationship between purchasing and price. Principal components analysis was used to examine the factor structure of RRE for food across samples and types of food. RESULTS: Both studies revealed two factor solutions, with Pmax, Omax, breakpoint and α loading on factor 1 (persistence) and intensity (Q0) loading on factor 2 (amplitude) across both high and low energy dense foods. Persistence reflects an aggregate measure of price sensitivity and amplitude reflects the preferred volume of consumption (how long vs. how much). The two factors accounted for between 91.7 and 95.4% of the variance in food reinforcement. Intensity for high energy dense foods predicted BMI for both studies (r = 0.18 and r = 0.22, p's < 0.05). CONCLUSIONS: The latent factor structure was similar across two significantly different independent samples and across low and high energy dense snack foods. In addition, the amplitude of the demand curve, but not persistence, was related to BMI. These results suggest specific aspects of food reinforcement that can be targeted to alter food intake.

Less is more: Negative income shock increases immediate preference in cross commodity discounting and food demand.

Negative income shock, or the rapid reduction in financial stability, has previously been shown to increase impulsive choice for money and demand for fast food. The interplay of these conditions for obesity is called reinforcer pathology. The present work examines the impact of negative income shock on monetary and fast food discounting using a cross-commodity delay discounting task and on purchasing of fast food and an alternative commodity. An obese sample (n = 120) was recruited from Amazon Mechanical Turk and assigned to read one of two narratives: negative income shock (n = 60) or control (n = 60). Participants then completed both within- and cross-commodity discounting tasks of money and food, and purchase tasks for fast food and bottled water. The negative income shock group demonstrated greater impulsive choice across discounting tasks, as well as higher intensity of demand for fast food but not for a non-caloric control commodity (bottled water). These results suggest that negative income shock increases preference for immediate reinforcement regardless of commodity type (money or fast food), but has specific effects increasing demand for particular commodities (fast food but not an alternative). In a reinforcer pathology framework, negative income shock increasing discounting of the future while increasing demand for fast food specifically represents a high-risk state for negative health behavior in obesity.

Hormonal responsiveness to stress is negatively associated with vulnerability to angling capture in fish.

Differences in behavior and physiology amongst individuals often alter relative fitness levels in the environment. However, the ideal behavioral/physiological phenotype in a given environment may be altered by human activity, leading to an evolutionary response in the affected population. One example of this process can be found in fisheries (including recreational freshwater fisheries), where selective capture and harvest of individuals with certain phenotypes can drive evolutionary change. While some life history traits and behavioral tendencies influencing capture likelihood have been studied, the physiological mechanisms driving this vulnerability remain poorly understood. To address this, we assessed how two major physiological characteristics (hormonal responsiveness to stress and metabolic phenotype) and one behavioral characteristic (boldness) impact the likelihood of an individual being captured by anglers. Largemouth bass, Micropterus salmoides, derived from a population artificially selected for differential angling vulnerability were assessed for boldness and for stress responsiveness (as indicated by plasma cortisol levels) following an air-exposure challenge. Largemouth bass were then stocked into a pond where experimental angling trials took place, and a subset of captured and uncaptured fish were afterwards assessed for metabolic phenotype. The results showed that stress responsiveness was the primary driver of angling vulnerability, with individuals that experienced lower rises in cortisol following the air-exposure challenge more likely to be captured. Neither boldness nor metabolic phenotype influenced capture probability. The results from this study indicate that fisheries-induced selective pressure may act on physiology, potentially altering stress responsiveness and its associated behaviors in populations exploited by recreational anglers.

Think fast: rapid assessment of the effects of episodic future thinking on delay discounting in overweight/obese participants.

Accumulating laboratory-based evidence indicates that reducing delay discounting (devaluation of delayed outcomes) with the use of episodic future thinking (EFT; mental simulation of future events) improves dietary decision-making and other maladaptive behaviors. Recent work has adapted EFT for use in the natural environment to aid in dietary and weight control by engaging participants in EFT repeatedly throughout the day. These efforts may benefit from minimizing the amount of time required for measurement and implementation of EFT. Using Amazon Mechanical Turk in the present study, we show that EFT effectively reduces delay discounting in overweight/obese participants (N = 131) using the recently developed 5-trial, adjusting-delay discounting task, which can be completed rapidly (25 s) and is therefore ideally suited for ecological momentary assessment. Moreover, measures of delay discounting from this task were strongly correlated with those from the commonly used adjusting-amount task (r = .859). Significant effects of EFT on discounting, however, depended on the number of future events participants generated and imagined. Use of a range of events and future time frames (as is typical in the literature) significantly reduced delay discounting, whereas use of only a single event did not.

Thrombospondin-1 differentially regulates microRNAs in vascular smooth muscle cells.

Thrombospondin-1 (TSP-1) is an important regulator of vascular smooth muscle cell (VSMC) physiology and gene expression. MicroRNAs (microRNA), small molecules that regulate protein translation, have emerged as potent regulators of cell function. MicroRNAs have been shown to be involved in intimal hyperplasia, atherosclerosis, and upregulated in the vasculature in diabetes. The purpose of this study was to identify microRNAs regulated by TSP-1 in vascular smooth muscle cells (VSMCs). Human VSMCs were treated for 6 h with basal media or TSP-1 both supplemented with 0.2% FBS. Cells were then snap frozen and RNA extracted. An Affymetrix GeneChip microRNA array analysis was performed in triplicate on three separate collections. Confirmatory qrtPCR was performed. Data were analyzed by ANOVA or t test, with significance set at p < 0.05. MicroRNAs identified were subjected to KEGG pathway analysis using the DIANA tools miRPath online tool. TSP-1 upregulated 22 microRNAs and downregulated 18 microRNAs in VSMCs (p < 0.05). The most upregulated microRNA was miR-512-3p (45.12 fold). The microRNA most downregulated by TSP-1 was miR-25-5p, which was decreased by 9.61. Of note, five members of the mir-17-92 cluster were downregulated. KEGG analysis revealed that thirty-three cellular signaling pathways were impacted by these microRNAs and that nine pathways were relevant to vascular disease. MicroRNAs regulate protein expression at the level of translation and may represent a significant mechanism by which TSP-1 regulates VSMC function. Several of the microRNAs identified have a role in vascular function. The miR-17-92 cluster family, which was found to exhibit reduced expression in this study, is known to be involved in angiogenesis and vascular function. TSP-1 regulates multiple microRNAs in VSMCs adding a new layer of complexity to TSP-1 regulation of VSMC function.

A key general stress response motif is regulated non-uniformly by CAMTA transcription factors.

Plants cope with environmental challenges by rapidly triggering and synchronizing mechanisms governing stress-specific and general stress response (GSR) networks. The GSR acts rapidly and transiently in response to various stresses, but the underpinning mechanisms have remained elusive. To define GSR regulatory components we have exploited the Rapid Stress Response Element (RSRE), a previously established functional GSR motif, using Arabidopsis plants expressing a 4xRSRE::Luciferase (RSRE::LUC) reporter. Initially, we searched public microarray datasets and found an enrichment of RSRE in promoter sequences of stress genes. Next, we treated RSRE::LUC plants with wounding and a range of rapidly stress-inducible hormones and detected a robust LUC activity solely in response to wounding. Application of two Ca(2+) burst inducers, flagellin22 (flg22) and oligogalacturonic acid, activated RSRE strongly and systemically, while the Ca(2+) chelator ethylene glycol tetraacetic acid (EGTA) significantly reduced wound induction of RSRE::LUC. In line with the signaling function of Ca(2+) in transduction events leading to activation of RSRE, we examined the role of CALMODULIN-BINDING TRANSCRIPTIONAL ACTIVATORs (CAMTAs) in RSRE induction. Transient expression assays displayed CAMTA3 induction of RSRE and not that of the mutated element mRSRE. Treatment of selected camta mutant lines integrated into RSRE::LUC parent plant, with wounding, flg22, and freezing, established a differential function of these CAMTAs in potentiating the activity of RSRE. Wound response studies using camta double mutants revealed a cooperative function of CAMTAs2 and 4 with CAMTA 3 in the RSRE regulation. These studies provide insights into governing components of transduction events and reveal transcriptional modules that tune the expression of a key GSR motif.