Crystal Structure of the Catalytic Domain of a Botulinum Neurotoxin Homologue from Enterococcus faecium: Potential Insights into Substrate Recognition.

Clostridium botulinum neurotoxins (BoNTs) are the most potent toxins known, causing the deadly disease botulism. They function through Zn2+-dependent endopeptidase cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, preventing vesicular fusion and subsequent neurotransmitter release from motor neurons. Several serotypes of BoNTs produced by Clostridium botulinum (BoNT/A-/G and/X) have been well-characterised over the years. However, a BoNT-like gene (homologue of BoNT) was recently identified in the non-clostridial species, Enterococcus faecium, which is the leading cause of hospital-acquired multi-drug resistant infections. Here, we report the crystal structure of the catalytic domain of a BoNT homologue from Enterococcus faecium (LC/En) at 2.0 Å resolution. Detailed structural analysis in comparison with the full-length BoNT/En AlphaFold2-predicted structure, LC/A (from BoNT/A), and LC/F (from BoNT/F) revealed putative subsites and exosites (including loops 1-5) involved in recognition of LC/En substrates. LC/En also appears to possess a conserved autoproteolytic cleavage site whose function is yet to be established.

Crystal Structures of the Clostridium botulinum Neurotoxin A6 Cell Binding Domain Alone and in Complex with GD1a Reveal Significant Conformational Flexibility.

Clostridium botulinum neurotoxin A (BoNT/A) targets the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, by cleaving synaptosomal-associated protein of 25 kDa size (SNAP-25). Cleavage of SNAP-25 results in flaccid paralysis due to repression of synaptic transmission at the neuromuscular junction. This activity has been exploited to treat a range of diseases associated with hypersecretion of neurotransmitters, with formulations of BoNT/A commercially available as therapeutics. Generally, BoNT activity is facilitated by three essential domains within the molecule, the cell binding domain (HC), the translocation domain (HN), and the catalytic domain (LC). The HC, which consists of an N-terminal (HCN) and a C-terminal (HCC) subdomain, is responsible for BoNT's high target specificity where it forms a dual-receptor complex with synaptic vesicle protein 2 (SV2) and a ganglioside receptor on the surface of motor neurons. In this study, we have determined the crystal structure of botulinum neurotoxin A6 cell binding domain (HC/A6) in complex with GD1a and describe the interactions involved in ganglioside binding. We also present a new crystal form of wild type HC/A6 (crystal form II) where a large 'hinge motion' between the HCN and HCC subdomains is observed. These structures, along with a comparison to the previously determined wild type crystal structure of HC/A6 (crystal form I), reveals the degree of conformational flexibility exhibited by HC/A6.

Assessing the Potential Impact of Cigarette Packs Designed for Lesbian, Gay, Bisexual, and Transgender Adults: A Randomized Experiment to Inform U.S. Regulation, 2018.

The Food and Drug Administration (FDA) can regulate the introduction of new tobacco products and some changes to existing products. Cigarette packs have been used as a marketing tool to target specific groups and priority populations. Research has shown that sexual and gender minority (SGM) adults are substantially more likely to use tobacco products than their straight and cisgender counterparts. However, research to inform the FDA's regulatory decisions regarding cigarette packs targeting priority populations is nascent. To fill this gap, we conducted an online experiment in 2018, randomizing U.S. adults who reported current smoking (N = 954, 52% were SGM) to view one of three cigarette packs. A graphic designer developed "Glacier" branded packs with three levels of SGM imagery: (1) no targeting, (2) subtle targeting, and (3) a rainbow "pride edition." Participants viewed and rated the pack using cognitive, affective, and behavioral measures informed by theory. We used a linear model framework to compare the two SGM-targeted packs with the not targeted version and tested interactions between pack and SGM identity for the dependent variables. We stratified results by SGM status. SGM status was a significant moderator of the relationship between the pack and ratings of appeal, positive affect, feeling shocked, and intent to try with a coupon. Findings from this study revealed that packs designed for SGM populations can disproportionately change cognitive, affective, and behavioral intention responses for SGM smokers. Products entering the market should be assessed by FDA for the appeal of their packs to vulnerable populations.

Evolving IQOS packaging designs change perceptions of product appeal, uniqueness, quality and safety: a randomised experiment, 2018, USA.

BACKGROUND: Globally, the tobacco industry is promoting heated tobacco products. These products may represent a strategy to promote dual use of tobacco products. One product, IQOS from Philip Morris International, is being proposed in the USA for marketing as a less harmful product. The visual design of tobacco products can influence consumers by implying product characteristics. Thus, we sought to test the impact of IQOS packaging designs on cognitive, affective and behavioural intention responses. METHODS: From existing IQOS packages used globally, we developed three IQOS packages that decreasingly linked the product to the Marlboro brand. In September to October 2018, we assigned participants randomly to one package in an online experiment. All participants (n=954) were US adults reporting current smoking and no colour blindness. The experiment used quota sampling to ensure diversity by gender, sexual orientation, race, ethnicity and education. Measures were informed by the Context of Consumption Framework. To assess differences in ratings, we conducted non-parametric Kruskal-Wallis tests with post hoc comparisons using Dunn's test. RESULTS: We found significant differences in cognitive indicators including appeal (H=6.87, p=0.03), uniqueness (H=15.68, p<0.01), brand equity-quality (H=122.35, p<0.01) and perceived safety compared with other tobacco products (H=14.27, p<0.01). Participants rated packages similarly on affective and behavioural intention measures. All were rated low for talking to others about the product and high for interest in trying with a coupon. CONCLUSION: Linking or separating IQOS products with a well-established cigarette brand changes how adult smokers respond to the product. Regulators should consider the visual design of packaging.

The Potential That Electronic Nicotine Delivery Systems Can be a Disruptive Technology: Results From a National Survey.

INTRODUCTION: This study evaluates the reasons for use and acceptance of Electronic Nicotine Delivery Systems (ENDS) among current and former cigarette smokers to assess if ENDS may become a satisfying alternative to cigarettes. METHODS: Data are from a national probability sample of 5717 US adults, surveyed June-November 2014. The survey contained questions on awareness, usage, and reasons for use of traditional and novel tobacco products. The analytic sample was current and former smokers who ever used ENDS (n = 729) and was divided into four mutually exclusive categories. Among the 585 current smokers, 337 were no longer using ENDS ("E-Cig Rejecters"), and 248 were continuing to use both ENDS and cigarettes ("E-Cig Dual Users"). Among 144 former cigarette smokers, 101 were non-recent users of ENDS ("Quit All Products"), and 43 were continuing to use ENDS exclusively ("Switchers"). RESULTS: Former smokers (the "Switchers") report finding ENDS a satisfying alternative to regular cigarettes, with only 15.8% (95% confidence interval [CI] 4.4-27.1) rating ENDS as less enjoyable than regular cigarettes. However, greater than fivefold more current smokers did not find them satisfying and stopped using them (77.3%; 95% CI 72.1-82.4 of "E-Cig Rejecters" rated ENDS as less enjoyable). Being less harmful was the most highly rated reason for continuing to use ENDS among "Switchers." Most (80.9%) "Switchers" reported that ENDS helped them quit cigarettes. CONCLUSION: Since many current smokers who have tried ENDS reject them as a satisfying alternative to regular cigarettes, ENDS will not replace regular cigarettes unless they improve. IMPLICATIONS: Since about one-half of recent former smokers are trying ENDS with about one-fourth continuing to use them, and many reporting that these products have helped them quit regular cigarettes, the potential impact of ENDS on population quit rates deserves continued surveillance. However, since most current smokers who have tried ENDS reject them as a satisfying alternative to regular cigarettes, the potential of ENDS becoming a disruptive technology replacing regular cigarettes remains uncertain. ENDS need to improve as a satisfying alternative or the attractiveness and appeal of the regular cigarette must be degraded to increase the potential of ENDS replacing regular cigarettes.

Inequities in tobacco retailer sales to minors by neighbourhood racial/ethnic composition, poverty and segregation, USA, 2015.

OBJECTIVE: Tobacco retailers are an important source of tobacco products for minors. Previous research shows racial discrimination in sales to minors, but no national study has examined neighbourhood correlates of retailer under-age sales. METHODS: We accessed publicly available results of 2015 US Food and Drug Administration (FDA) inspections of tobacco retailers (n=108 614). In this cross-sectional study, we used multilevel logistic regression to predict the likelihood of retailer sale to a minor based on tract characteristics. We assessed the proportion of residents identifying as American Indian, Asian, Black, Latino and White; Isolation Index scores for each racial/ethnic group; the proportion of people less than age 65 living in poverty; and the proportion of residents age 10-17 in relation to retailer inspection results. RESULTS: The proportion of American Indian residents, Black residents, Latino residents and residents less than age 65 under the poverty line in a neighbourhood are independently, positively associated with the likelihood that a retailer in that neighbourhood will fail an under-age buy inspection. The proportion of White residents and residents age 10-17 are independently, negatively associated with the likelihood of sale of tobacco products to a minor. Isolation Index scores show a similar pattern. In multivariable models holding neighbourhood characteristics constant, higher proportions of Black (+), Latino (+) and age 10-17 (-) residents remained significant predictors of the likelihood of under-age sale. DISCUSSION: Regulatory agencies should consider oversampling retailers in areas with higher likelihood of sales to minors for inspection. Interventions with tobacco retailers to reduce inequities in youth access should be implemented.

Functional implications of unusual NOS and SONOS covalent linkages found in proteins.

The tertiary and quaternary structures of many proteins are stabilized by strong covalent forces, of which disulfide bonds are the most well known. A new type of intramolecular and intermolecular covalent bond has been recently reported, consisting of the Lys and Cys side-chains linked by an oxygen atom (NOS). These post-translational modifications are widely distributed amongst proteins, and are formed under oxidative conditions. Similar linkages are observed during antibiotic biosynthesis, where hydroxylamine intermediates are tethered to the sulfur of enzyme active site Cys residues. These linkages open the way to understanding protein structure and function, give new insights into enzyme catalysis and natural product biosynthesis, and offer new strategies for drug design.

Advances in the structural basis for angiotensin-1 converting enzyme (ACE) inhibitors.

Human somatic angiotensin-converting enzyme (ACE) is a key zinc metallopeptidase that plays a pivotal role in the renin-angiotensin-aldosterone system (RAAS) by regulating blood pressure and electrolyte balance. Inhibition of ACE is a cornerstone in the management of hypertension, cardiovascular diseases, and renal disorders. Recent advances in structural biology techniques have provided invaluable insights into the molecular mechanisms underlying ACE inhibition, facilitating the design and development of more effective therapeutic agents. This review focuses on the latest advancements in elucidating the structural basis for ACE inhibition. High-resolution crystallographic studies of minimally glycosylated individual domains of ACE have revealed intricate molecular details of the ACE catalytic N- and C-domains, and their detailed interactions with clinically relevant and newly designed domain-specific inhibitors. In addition, the recently elucidated structure of the glycosylated form of full-length ACE by cryo-electron microscopy (cryo-EM) has shed light on the mechanism of ACE dimerization and revealed continuous conformational changes which occur prior to ligand binding. In addition to these experimental techniques, computational approaches have also played a pivotal role in elucidating the structural basis for ACE inhibition. Molecular dynamics simulations and computational docking studies have provided atomic details of inhibitor binding kinetics and energetics, facilitating the rational design of novel ACE inhibitors with improved potency and selectivity. Furthermore, computational analysis of the motions observed by cryo-EM allowed the identification of allosteric binding sites on ACE. This affords new opportunities for the development of next-generation allosteric inhibitors with enhanced pharmacological properties. Overall, the insights highlighted in this review could enable the rational design of novel ACE inhibitors with improved efficacy and safety profiles, ultimately leading to better therapeutic outcomes for patients with hypertension and cardiovascular diseases.

Structural insights into the inhibitory mechanism of angiotensin-I-converting enzyme by the lactotripeptides IPP and VPP.

Human somatic angiotensin-1-converting enzyme (sACE) is composed of a catalytic N-(nACE) and C-domain (cACE) of similar size with different substrate specificities. It is involved in the regulation of blood pressure by converting angiotensin I to the vasoconstrictor angiotensin II and has been a major focus in the development of therapeutics for hypertension. Bioactive peptides from various sources, including milk, have been identified as natural ACE inhibitors. We report the structural basis for the role of two lacototripeptides, Val-Pro-Pro and Ile-Pro-Pro, in domain-specific inhibition of ACE using X-ray crystallography and kinetic analysis. The lactotripeptides have preference for nACE due to altered polar interactions distal to the catalytic zinc ion. Elucidating the mechanism of binding and domain selectivity of these peptides also provides important insights into the functional roles of ACE.

A Comprehensive Structural Analysis of Clostridium botulinum Neurotoxin A Cell-Binding Domain from Different Subtypes.

Botulinum neurotoxins (BoNTs) cause flaccid neuromuscular paralysis by cleaving one of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex proteins. BoNTs display high affinity and specificity for neuromuscular junctions, making them one of the most potent neurotoxins known to date. There are seven serologically distinct BoNTs (serotypes BoNT/A to BoNT/G) which can be further divided into subtypes (e.g., BoNT/A1, BoNT/A2…) based on small changes in their amino acid sequence. Of these, BoNT/A1 and BoNT/B1 have been utilised to treat various diseases associated with spasticity and hypersecretion. There are potentially many more BoNT variants with differing toxicological profiles that may display other therapeutic benefits. This review is focused on the structural analysis of the cell-binding domain from BoNT/A1 to BoNT/A6 subtypes (HC/A1 to HC/A6), including features such as a ganglioside binding site (GBS), a dynamic loop, a synaptic vesicle glycoprotein 2 (SV2) binding site, a possible Lys-Cys/Cys-Cys bridge, and a hinge motion between the HCN and HCC subdomains. Characterising structural features across subtypes provides a better understanding of how the cell-binding domain functions and may aid the development of novel therapeutics.

A Molecular Analysis of the Aminopeptidase P-Related Domain of PID-5 from Caenorhabditis elegans.

A novel protein, PID-5, has been shown to be a requirement for germline immortality and has recently been implicated in RNA-induced epigenetic silencing in the Caenorhabditis elegans embryo. Importantly, it has been shown to contain both an eTudor and aminopeptidase P-related domain. However, the silencing mechanism has not yet been fully characterised. In this study, bioinformatic tools were used to compare pre-existing aminopeptidase P molecular structures to the AlphaFold2-predicted aminopeptidase P-related domain of PID-5 (PID-5 APP-RD). Structural homology, metal composition, inhibitor-bonding interactions, and the potential for dimerisation were critically assessed through computational techniques, including structural superimposition and protein-ligand docking. Results from this research suggest that the metallopeptidase-like domain shares high structural homology with known aminopeptidase P enzymes and possesses the canonical 'pita-bread fold'. However, the absence of conserved metal-coordinating residues indicates that only a single Zn2+ may be bound at the active site. The PID-5 APP-RD may form transient interactions with a known aminopeptidase P inhibitor and may therefore recognise substrates in a comparable way to the known structures. However, loss of key catalytic residues suggests the domain will be inactive. Further evidence suggests that heterodimerisation with C. elegans aminopeptidase P is feasible and therefore PID-5 is predicted to regulate proteolytic cleavage in the silencing pathway. PID-5 may interact with PID-2 to bring aminopeptidase P activity to the Z-granule, where it could influence WAGO-4 activity to ensure the balanced production of 22G-RNA signals for transgenerational silencing. Targeted experiments into APPs implicated in malaria and cancer are required in order to build upon the biological and therapeutic significance of this research.

Structural Features of Clostridium botulinum Neurotoxin Subtype A2 Cell Binding Domain.

Botulinum neurotoxins (BoNT) are a group of clostridial toxins that cause the potentially fatal neuroparalytic disease botulism. Although highly toxic, BoNTs are utilized as therapeutics to treat a range of neuromuscular conditions. Several serotypes (BoNT/A-/G, /X) have been identified with vastly differing toxicological profiles. Each serotype can be further sub-categorised into subtypes due to subtle variations in their protein sequence. These minor changes have been attributed to differences in both the duration of action and potency for BoNT/A subtypes. BoNTs are composed of three domains-a cell-binding domain, a translocation domain, and a catalytic domain. In this paper, we present the crystal structures of the botulinum neurotoxin A2 cell binding domain, both alone and in complex with its receptor ganglioside GD1a at 1.63 and 2.10 Å, respectively. The analysis of these structures reveals a potential redox-dependent Lys-O-Cys bridge close to the ganglioside binding site and a hinge motion between the HCN and HCC subdomains. Furthermore, we make a detailed comparison with the previously reported HC/A2:SV2C structure for a comprehensive structural analysis of HC/A2 receptor binding.

Crystal Structures of Botulinum Neurotoxin Subtypes A4 and A5 Cell Binding Domains in Complex with Receptor Ganglioside.

Botulinum neurotoxins (BoNT) cause the potentially fatal neuroparalytic disease botulism that arises due to proteolysis of a SNARE protein. Each BoNT is comprised of three domains: a cell binding domain (HC), a translocation domain (HN), and a catalytic (Zn2+ endopeptidase) domain (LC). The HC is responsible for neuronal specificity by targeting both a protein and ganglioside receptor at the neuromuscular junction. Although highly toxic, some BoNTs are commercially available as therapeutics for the treatment of a range of neuromuscular conditions. Here we present the crystal structures of two BoNT cell binding domains, HC/A4 and HC/A5, in a complex with the oligosaccharide of ganglioside, GD1a and GM1b, respectively. These structures, along with a detailed comparison with the previously reported apo-structures, reveal the conformational changes that occur upon ganglioside binding and the interactions involved.

Developmental changes in cardiac expression of KCNQ1 and SCN5A spliceoforms: Implications for sudden unexpected infant death.

BACKGROUND: Sudden unexpected infant death (SUID) occurs unpredictably and remains unexplained after scene investigation and autopsy. Approximately 1 in 7 cases of SUID can be related to a cardiac cause, and developmental regulation of cardiac ion channel genes may contribute to SUID. OBJECTIVE: The goal of this study was to investigate the developmental changes in the spliceoforms of SCN5A and KCNQ1, 2 genes implicated in SUID. METHODS: Using reverse transcription quantitative real-time polymerase chain reaction, we quantified expression of SCN5A (adult and fetal) and KCNQ1 (KCNQ1a and b) spliceoforms in 153 human cardiac tissue samples from decedents that succumbed to SUID ("unexplained") and other known causes of death ("explained noncardiac"). RESULTS: There is a stepwise increase in the adult/fetal SCN5A spliceoform ratio from <2 months (4.55 ± 0.36; n = 51) through infancy and into adulthood (17.41 ± 3.33; n = 5). For KCNQ1, there is a decrease in the ratio of KCNQ1b to KCNQ1a between the <2-month (0.37 ± 0.02; n = 46) and the 2- to 4-month (0.28 ± 0.02; n = 52) age groups. When broken down by sex, race, or cause of death, there were no differences in SCN5A or KCNQ1 spliceoform expression, except for a higher ratio of KCNQ1b to KCNQ1a at 5-12 months of age for SUID females (0.40 ± 0.04; n = 9) than for males (0.25 ± 0.03; n = 6) and at <2 months of age for SUID white (0.42 ± 0.03; n = 19) than for black (0.33 ± 0.05; n = 9) infants. CONCLUSION: This study documents the developmental changes in SCN5A and KCNQ1 spliceoforms in humans. Our data suggest that spliceoform expression ratios change significantly throughout the first year of life.

Changes to cigarette packaging influence US consumers' choices: Results of two discrete-choice experiments to inform regulation.

INTRODUCTION: While plain packaging of tobacco products has emerged as a policy intervention to reduce smoking, regulators in the US have limited ability to implement plain packaging. We sought to identify the impact of subtle changes to cigarette packaging (Study 1) and how packaging design influenced participant choices based on appeal, harm, and style (Study 2). METHODS: We conducted two discrete-choice experiments with US adult smokers online in 2018. In Study 1 (n=285), we assessed participants' selections based on subtle changes to pack design features (dimensions, color saturation, logo size). In Study 2 (n=284), we assessed three choices in which participants selected packs based on appeal, harmfulness, and best match to their personal style. Study 2 packs varied by color hue, design with different levels of organic labeling and natural imagery, and color saturation. RESULTS: Pack designs influenced smokers' choices. In Study 1, pack dimensions and color saturation emerged as the most important features, and, in Study 2, design and color hue were the most influential characteristics. CONCLUSIONS: Regulators should consider how the design of cigarette packages may influence consumers' perceptions and choices.

Crystal structure of botulinum neurotoxin subtype A3 cell binding domain in complex with GD1a co-receptor ganglioside.

Botulinum neurotoxins (BoNTs) are one of the most toxic proteins known to humans. Their molecular structure is comprised of three essential domains-a cell binding domain (HC ), translocation domain and catalytic domain (light chain) . The HC domain facilitates the highly specific binding of BoNTs to the neuronal membrane via a dual-receptor complex involving a protein receptor and a ganglioside. Variation in activity/toxicity across subtypes of serotype A has been attributed to changes in protein and ganglioside interactions, and their implications are important in the design of novel BoNT-based therapeutics. Here, we present the structure of BoNT/A3 cell binding domain (HC /A3) in complex with the ganglioside GD1a at 1.75 Å resolution. The structure revealed that six residues interact with the three outermost monosaccharides of GD1a through several key hydrogen bonding interactions. A detailed comparison of structures of HC /A3 with HC /A1 revealed subtle conformational differences at the ganglioside binding site upon carbohydrate binding.

"The Packaging Is Very Inviting and Makes Smokers Feel Like They're More Safe": The Meanings of Natural American Spirit Cigarette Pack Design to Adult Smokers.

BACKGROUND/AIMS: The aim of this investigation was to identify which design elements on Natural American Spirit packs are salient to (i.e., noticed by) U.S. adult smokers and what meanings smokers derive from these elements. METHOD: We conducted a secondary analysis of qualitative data from a study of cigarette packaging design. U.S. adult smokers ( n = 33) from all nine census regions participated in six telephone-based focus groups in March 2017. We used constant comparison analysis to identify key themes. RESULTS: Four themes were identified, two focused on salient design elements and two focused on design element meanings. The themes of "bright and flashy color" and "the American Indian logo" were identified as key design elements, while the themes of "healthy and safer" and "targeting at-risk smokers" were identified as meanings smokers derived from design elements. CONCLUSIONS: Pack design elements influence smokers' perceptions about reduced health risk of Natural American Spirit cigarettes and may be especially dangerous to vulnerable populations, including young adults and American Indians. Findings from this study suggest that the banning of text descriptors may not be enough to address misconceptions about "healthier" cigarettes.

Is the cigarette pack just a wrapper or a characteristic of the product itself? A qualitative study of adult smokers to inform U.S. regulations.

PURPOSE: In the U.S., tobacco products are now regulated by the Food and Drug Administration (FDA). Litigation has quickly followed. One area of controversy is when a change to the design of the cigarette pack requires approval through FDA's rigorous premarket review process. In this paper, we examine how adult U.S. smokers view the connection between the design of cigarette packs and the characteristics of the cigarettes within. METHODS: Data for this qualitative study came from six focus groups conducted in March 2017 with adult smokers. Two groups consisted of lesbian, gay, and bisexual (LGB) participants; two groups of participants with less than four years of college education; one group of LGB and straight identity; and, one group of the general population. All groups were selected for regional, gender, and racial/ethnic diversity. Participants (n = 33) represented all nine U.S. Census divisions. We conducted constant comparison qualitative analysis utilizing a grounded theory approach. RESULTS: Participants' views reflected a belief that pack design is clearly a reflection of the cigarettes within and that a change in the pack signaled a change in the cigarettes. However, some participants felt price was the salient characteristic of cigarettes and design mattered more for enticing young people to smoke. CONCLUSIONS: Changes in pack design signal changes to the product for smokers. Pack design and changes to pack design are seen as particularly relevant to new and young smokers. These findings provide support for regulations that require assessment of cigarette pack design changes for impacts on public health.

Qualitative assessment of a Context of Consumption Framework to inform regulation of cigarette pack design in the U.S.

INTRODUCTION: Researchers and regulators need to know how changes to cigarette packages can influence population health. We sought to advance research on the role of cigarette packaging by assessing a theory-informed framework from the fields of design and consumer research. The selected Context of Consumption Framework posits cognitive, affective, and behavioral responses to visual design. To assess the Framework's potential for guiding research on the visual design of cigarette packaging in the U.S., this study seeks to understand to what extent the Context of Consumption Framework converges with how adult smokers think and talk about cigarette pack designs. METHODS: Data for this qualitative study came from six telephone-based focus groups conducted in March 2017. Two groups consisted of lesbian, gay, and bisexual participants; two groups of participants with less than four years college education; one group of LGB and straight identity; and one group the general population. All groups were selected for regional, gender, and racial/ethnic diversity. Participants (n=33) represented all nine U.S. Census divisions. We conducted a deductive qualitative analysis. RESULTS: Cigarette package designs captured the participants' attention, suggested the characteristics of the product, and reflected (or could be leveraged to convey) multiple dimensions of consumer identity. Particular to the affective responses to design, our participants shared that cigarette packaging conveyed how the pack could be used to particular ends, created an emotional response to the designs, complied with normative expectations of a cigarette, elicited interest when designs change, and prompted fascination when unique design characteristics are used. CONCLUSIONS: Use of the Context of Consumption Framework for cigarette product packaging design can inform regulatory research on tobacco product packaging. Researchers and regulators should consider multiple cognitive, affective, and behavioral responses to cigarette pack design.

"Their Packaging Has Always Been Like a Power": A Qualitative Study of U.S. Smokers' Perceptions of Cigarette Pack Visual Design Features to Inform Product Regulation.

Cigarette packaging matters to consumer behavior. However, it is less clear which changes to packaging design would be salient for adult smokers. Such information is critically important to regulators in the United States who are charged with reviewing new tobacco products for their impact on population health. In this qualitative study, U.S. adult smokers (n = 33) participated in six telephone-based focus groups in March 2017. Separate groups were comprised of lesbian, gay, and bisexual (LGB) participants; participants with less than four years of post-secondary education; a mix of LGB and straight participants; and, the general population. All groups were purposely selected for diversity. Open thematic coding identified salient design elements used on cigarette packaging. Smokers articulated design elements' use, meaning, and links with consumer behaviors. Three themes were identified: (1) the power of color, (2) supporting color with other design elements (e.g., logos/images, typography, the pack itself), and (3) the combined product brand experience of multiple design elements. Participants linked design elements to product characteristics and to consumer behavior (e.g., purchase). As the Food and Drug Administration is charged with regulating tobacco products, these findings suggest the importance of considering the cigarette pack part of the characteristics of a product.