Critical Role for the DNA Sensor AIM2 in Stem Cell Proliferation and Cancer.

Colorectal cancer is a leading cause of cancer-related deaths. Mutations in the innate immune sensor AIM2 are frequently identified in patients with colorectal cancer, but how AIM2 modulates colonic tumorigenesis is unknown. Here, we found that Aim2-deficient mice were hypersusceptible to colonic tumor development. Production of inflammasome-associated cytokines and other inflammatory mediators was largely intact in Aim2-deficient mice; however, intestinal stem cells were prone to uncontrolled proliferation. Aberrant Wnt signaling expanded a population of tumor-initiating stem cells in the absence of AIM2. Susceptibility of Aim2-deficient mice to colorectal tumorigenesis was enhanced by a dysbiotic gut microbiota, which was reduced by reciprocal exchange of gut microbiota with healthy wild-type mice. These findings uncover a synergy between a specific host genetic factor and gut microbiota in determining the susceptibility to colorectal cancer. Therapeutic modulation of AIM2 expression and microbiota has the potential to prevent colorectal cancer.

Lung tissue shows divergent gene expression between chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are characterized by shared exposures and clinical features, but distinct genetic and pathologic features exist. These features have not been well-studied using large-scale gene expression datasets. We hypothesized that there are divergent gene, pathway, and cellular signatures between COPD and IPF. METHODS: We performed RNA-sequencing on lung tissues from individuals with IPF (n = 231) and COPD (n = 377) compared to control (n = 267), defined as individuals with normal spirometry. We grouped the overlapping differential expression gene sets based on direction of expression and examined the resultant sets for genes of interest, pathway enrichment, and cell composition. Using gene set variation analysis, we validated the overlap group gene sets in independent COPD and IPF data sets. RESULTS: We found 5010 genes differentially expressed between COPD and control, and 11,454 genes differentially expressed between IPF and control (1% false discovery rate). 3846 genes overlapped between IPF and COPD. Several pathways were enriched for genes upregulated in COPD and downregulated in IPF; however, no pathways were enriched for genes downregulated in COPD and upregulated in IPF. There were many myeloid cell genes with increased expression in COPD but decreased in IPF. We found that the genes upregulated in COPD but downregulated in IPF were associated with lower lung function in the independent validation cohorts. CONCLUSIONS: We identified a divergent gene expression signature between COPD and IPF, with increased expression in COPD and decreased in IPF. This signature is associated with worse lung function in both COPD and IPF.

Exome sequencing identifies genetic variants in anophthalmia and microphthalmia.

Anophthalmia and microphthalmia (A/M) are rare birth defects affecting up to 2 per 10,000 live births. These conditions are manifested by the absence of an eye or reduced eye volumes within the orbit leading to vision loss. Although clinical case series suggest a strong genetic component in A/M, few systematic investigations have been conducted on potential genetic contributions owing to low population prevalence. To overcome this challenge, we utilized DNA samples and data collected as part of the National Birth Defects Prevention Study (NBDPS). The NBDPS employed multi-center ascertainment of infants affected by A/M. We performed exome sequencing on 67 family trios and identified numerous genes affected by rare deleterious nonsense and missense variants in this cohort, including de novo variants. We identified 9 nonsense changes and 86 missense variants that are absent from the reference human population (Genome Aggregation Database), and we suggest that these are high priority candidate genes for A/M. We also performed literature curation, single cell transcriptome comparisons, and molecular pathway analysis on the candidate genes and performed protein structure modeling to determine the potential pathogenic variant consequences on PAX6 in this disease.

Exome sequencing of child-parent trios with bladder exstrophy: Findings in 26 children.

Bladder exstrophy (BE) is a rare, lower ventral midline defect with the bladder and part of the urethra exposed. The etiology of BE is unknown but thought to be influenced by genetic variation with more recent studies suggesting a role for rare variants. As such, we conducted paired-end exome sequencing in 26 child/mother/father trios. Three children had rare (allele frequency ≤ 0.0001 in several public databases) inherited variants in TSPAN4, one with a loss-of-function variant and two with missense variants. Two children had loss-of-function variants in TUBE1. Four children had rare missense or nonsense variants (one per child) in WNT3, CRKL, MYH9, or LZTR1, genes previously associated with BE. We detected 17 de novo missense variants in 13 children and three de novo loss-of-function variants (AKR1C2, PRRX1, PPM1D) in three children (one per child). We also detected rare compound heterozygous loss-of-function variants in PLCH2 and CLEC4M and rare inherited missense or loss-of-function variants in additional genes applying autosomal recessive (three genes) and X-linked recessive inheritance models (13 genes). Variants in two genes identified may implicate disruption in cell migration (TUBE1) and adhesion (TSPAN4) processes, mechanisms proposed for BE, and provide additional evidence for rare variants in the development of this defect.

Dietary modulation of the microbiome affects autoinflammatory disease.

The incidences of chronic inflammatory disorders have increased considerably over the past three decades. Recent shifts in dietary consumption may have contributed importantly to this surge, but how dietary consumption modulates inflammatory disease is poorly defined. Pstpip2(cmo) mice, which express a homozygous Leu98Pro missense mutation in the Pombe Cdc15 homology family protein PSTPIP2 (proline-serine-threonine phosphatase interacting protein 2), spontaneously develop osteomyelitis that resembles chronic recurrent multifocal osteomyelitis in humans. Recent reports demonstrated a crucial role for interleukin-1ß (IL-1ß) in osteomyelitis, but deletion of the inflammasome components caspase-1 and NLRP3 failed to rescue Pstpip2(cmo) mice from inflammatory bone disease. Thus, the upstream mechanisms controlling IL-1ß production in Pstpip2(cmo) mice remain to be identified. In addition, the environmental factors driving IL-1ß-dependent inflammatory bone erosion are unknown. Here we show that the intestinal microbiota of diseased Pstpip2(cmo) mice was characterized by an outgrowth of Prevotella. Notably, Pstpip2(cmo) mice that were fed a diet rich in fat and cholesterol maintained a normal body weight, but were markedly protected against inflammatory bone disease and bone erosion. Diet-induced protection against osteomyelitis was accompanied by marked reductions in intestinal Prevotella levels and significantly reduced pro-IL-1ß expression in distant neutrophils. Furthermore, pro-IL-1ß expression was also decreased in Pstpip2(cmo) mice treated with antibiotics, and in wild-type mice that were kept under germ-free conditions. We further demonstrate that combined deletion of caspases 1 and 8 was required for protection against IL-1ß-dependent inflammatory bone disease, whereas the deletion of either caspase alone or of elastase or neutrophil proteinase 3 failed to prevent inflammatory disease. Collectively, this work reveals diet-associated changes in the intestinal microbiome as a crucial factor regulating inflammasome- and caspase-8-mediated maturation of IL-1ß and osteomyelitis in Pstpip2(cmo) mice.

The genetic basis of early T-cell precursor acute lymphoblastic leukaemia.

Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.

Genome-wide and single-cell analyses reveal a context dependent relationship between CBP recruitment and gene expression.

Genome-wide distribution of histone H3K18 and H3K27 acetyltransferases, CBP (CREBBP) and p300 (EP300), is used to map enhancers and promoters, but whether these elements functionally require CBP/p300 remains largely uncertain. Here we compared global CBP recruitment with gene expression in wild-type and CBP/p300 double-knockout (dKO) fibroblasts. ChIP-seq using CBP-null cells as a control revealed nearby CBP recruitment for 20% of constitutively-expressed genes, but surprisingly, three-quarters of these genes were unaffected or slightly activated in dKO cells. Computationally defined enhancer-promoter-units (EPUs) having a CBP peak near the enhancer-like element were more predictive, with CBP/p300 deletion attenuating expression of 40% of such constitutively-expressed genes. Examining signal-responsive (Hypoxia Inducible Factor) genes showed that 97% were within 50 kilobases of an inducible CBP peak, and 70% of these required CBP/p300 for full induction. Unexpectedly, most inducible CBP peaks occurred near signal-nonresponsive genes. Finally, single-cell expression analysis revealed additional context dependence where some signal-responsive genes were not uniformly dependent on CBP/p300 in individual cells. While CBP/p300 was needed for full induction of some genes in single-cells, for other genes CBP/p300 increased the probability of maximal expression. Thus, target gene context influences the transcriptional requirement for CBP/p300, possibly by multiple mechanisms.

Control of virulence by small RNAs in Streptococcus pneumoniae.

Small noncoding RNAs (sRNAs) play important roles in gene regulation in both prokaryotes and eukaryotes. Thus far, no sRNA has been assigned a definitive role in virulence in the major human pathogen Streptococcus pneumoniae. Based on the potential coding capacity of intergenic regions, we hypothesized that the pneumococcus produces many sRNAs and that they would play an important role in pathogenesis. We describe the application of whole-genome transcriptional sequencing to systematically identify the sRNAs of Streptococcus pneumoniae. Using this approach, we have identified 89 putative sRNAs, 56 of which are newly identified. Furthermore, using targeted genetic approaches and Tn-seq transposon screening, we demonstrate that many of the identified sRNAs have important global and niche-specific roles in virulence. These data constitute the most comprehensive analysis of pneumococcal sRNAs and provide the first evidence of the extensive roles of sRNAs in pneumococcal pathogenesis.

Social Interest Data as a Proxy for Off-Label Performance-Enhancing Drug Use: Implications and Clinical Considerations.

Performance-enhancing drugs (PEDs) can be categorized into various classes based on the physiological mechanism of the compound, with the most popular being anabolic steroids, selective androgen receptor modulators, and growth hormones. Ancillary compounds, such as selective estrogen receptor modulators (SERMs) and selective estrogen receptor degraders, are commonly utilized alongside a PED to counterbalance any potential undesired side effects. With little clinically relevant data to support the use of these ancillary compounds, medical education and evidence-based approaches aimed at monitoring the potential adverse effects of PED use are sparse.This study aims to identify emerging trends in the interest of PEDs and related ancillary compounds, hypothesize the physiological effects of the continued respective behavior, and propose a proxy for use by clinicians to approximate off-label drug use and subsequently modify their practices accordingly. Several significant trends were identified for non-FDA-regulated compounds (i.e., selective androgen receptor modulators such as RAD-140) and off-label indications for FDA-regulated drugs (i.e., SERMs such as tamoxifen). A significant increase in interest regarding selective androgen receptor modulators, mirrored by anecdotal reports in clinical settings and online forums, is coupled with stagnant or decreasing interest in both post-cycle therapies and anabolic steroids. Ultimately, we propose a call to action for utilizing social data and/or prescription data as a proxy for clinicians to better understand trends in these compounds and thus refine their treatment protocols in a concordant manner.

Ordered progression of stage-specific miRNA profiles in the mouse B2 B-cell lineage.

Micro-RNAs (miRNAs) have been recognized as critical regulators of gene expression, and deregulation of miRNA expression has been implicated in a wide spectrum of diseases. To provide a framework for the role of miRNAs in B-cell development and malignancy, we deep-sequenced miRNAs from B1 cells and 10 developmental stages that can be identified within the mouse B2 B-cell lineage. The expression profiles of the 232 known miRNAs that are expressed during B-cell development display stage-specific induction patterns, yet hierarchical clustering analysis showed relationships that are in full agreement with the model of the B2 B-cell developmental pathway. Analysis of exemplary miRNA expression profiles (miR-150, miR-146a, miR-155, miR-181) confirmed that our data are in agreement with previous results. The high resolution of the expression data allowed for the identification of the sequential expression of oncomir-1/miR-17-92 and its paralogs miR-106a-363 and miR-106b-25 in subsequent developmental stages in the BM. Further, we have identified and validated 45 novel miRNAs and 6 novel miRNA candidates expressed in developing B cells.

Extracellular Matrix Secretion Mechanically Reinforces Interlocking Interfaces.

Drawing inspiration for biomaterials from biological systems has led to many biomedical innovations. One notable bioinspired device, Velcro, consists of two substrates with interlocking ability. Generating reversibly interlocking biomaterials is an area of investigation, as such devices can allow for modular tissue engineering, reversibly interlocking biomaterial interfaces, or friction-based coupling devices. Here, a biaxially interlocking interface generated using electrostatic flocking is reported. Two electrostatically flocked substrates are mechanically and reversibly interlocked with the ability to resist shearing and compression forces. An initial high-throughput screen of polyamide flock fibers with varying diameters and fiber lengths is conducted to elucidate the roles of different fiber parameters on scaffold mechanical properties. After determining the most desirable parameters via weight scoring, polylactic acid (PLA) fibers are used to emulate the ideal scaffold for in vitro use. PLA flocked scaffolds are populated with osteoblasts and interlocked. Interlocked flocked scaffolds improved cell survivorship under mechanical compression and sustained cell viability and proliferation. Additionally, the compression and shearing resistance of cell-seeded interlocking interfaces increased with increasing extracellular matrix deposition. The introduction of extracellular matrix-reinforced interlocking interfaces may serve as binders for modular tissue engineering, act as scaffolds for engineering tissue interfaces, or enable friction-based couplers for biomedical applications.

Exome sequencing identifies variants in infants with sacral agenesis.

BACKGROUND: Sacral agenesis (SA) consists of partial or complete absence of the caudal end of the spine and often presents with additional birth defects. Several studies have examined gene variants for syndromic forms of SA, but only one has examined exomes of children with non-syndromic SA. METHODS: Using buccal cell specimens from families of children with non-syndromic SA, exomes of 28 child-parent trios (eight with and 20 without a maternal diagnosis of pregestational diabetes) and two child-father duos (neither with diagnosis of maternal pregestational diabetes) were exome sequenced. RESULTS: Three children had heterozygous missense variants in ID1 (Inhibitor of DNA Binding 1), with CADD scores >20 (top 1% of deleterious variants in the genome); two children inherited the variant from their fathers and one from the child's mother. Rare missense variants were also detected in PDZD2 (PDZ Domain Containing 2; N = 1) and SPTBN5 (Spectrin Beta, Non-erythrocytic 5; N = 2), two genes previously suggested to be associated with SA etiology. Examination of variants with autosomal recessive and X-linked recessive inheritance identified five and two missense variants, respectively. Compound heterozygous variants were identified in several genes. In addition, 12 de novo variants were identified, all in different genes in different children. CONCLUSIONS: To our knowledge, this is the first study reporting a possible association between ID1 and non-syndromic SA. Although maternal pregestational diabetes has been strongly associated with SA, the missense variants in ID1 identified in two of three children were paternally inherited. These findings add to the knowledge of gene variants associated with non-syndromic SA and provide data for future studies.

Analyzing Public Interest in Metabolic Health-Related Search Terms During COVID-19 Using Google Trends.

Background In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a novel virus and initiated a series of events that culminated in the global coronavirus disease 2019 (COVID-19) pandemic. Throughout 2020 and the first half of 2021, massive investigational efforts towards identifying, treating, preventing, and slowing the spread of COVID-19 were carried out. Several predictors for clinical outcomes relating to metabolic health were identified. Aim and methods This study aimed to investigate how public interest in search terms associated with metabolic health has changed throughout and during the course of the COVID-19 pandemic. Google Trends was utilized as a tool to gather and compare public interest data in a variety of search phrases. The relative search values were plotted over time, compared pre-and post-COVID-19, analyzed for correlation, assessed for trend directionality, and checked for trend inclusion. Results The public interest measured by relative search volume in "metabolic health," "exercise," "home exercise," "health," and "how to improve fitness" significantly increased from pre- to post-COVID-19 pandemic onset while "diet" and "fitness" significantly decreased. The search terms "COVID" and "coronavirus" made up more than 95% of screen queries incorporating COVID-19. During the COVID-19 pandemic, "diabetes" and "weight loss" had the most significant increases in search volume. Conclusions Given the changes in public interest observed throughout the course of the COVID-19 pandemic, it is clear that the association between metabolic health and COVID-19 is being successfully disseminated to the public. However, these changes also warrant increased public education surrounding diet and fitness to align public interest with measures proven to improve the clinical outcomes of COVID-19.

Social Data: An Underutilized Metric for Determining Participation in COVID-19 Vaccinations.

Many measures have been taken since late 2019 to combat the coronavirus disease (COVID-19) pandemic. National, state, and local governments employed precautions, including mask mandates, stay-at-home orders, and social distancing policies, to alleviate the burden on healthcare workers and slow the spread of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) virus until an efficacious vaccine was made widely available. By early spring of 2021, three effective and well-tolerated SARS-CoV-2 vaccines emerged and underwent broad distribution. Throughout the course of the COVID-19 vaccination campaign, several key logistical and psychological issues surfaced. Of these, access to vaccines and vaccination hesitancy are cited as two substantial hindrances towards vaccination. Noting the demand for the SARS-CoV-2 vaccine and its highly sensitive storage requirements, accurate dose allocation is critical for vaccinating the population quickly and successfully. Here, we propose the use of social data as a tool to predict vaccination participation by correlating Google searches with state-level daily vaccination. We identified a temporal and regionally-ubiquitous Google search syntax that broadly captures daily vaccination trends. By correlating trends in the search syntax with daily vaccination rates, we were able to quantify the correlation and identify optimal lag periods between Google searches and daily vaccination. This work highlights the importance of analyzing social data as a metric to effectively arrange vaccination roll-outs, identify voluntary vaccination participation, and identify inflection points in vaccination participation. In addition, social data assessments can help direct dose allocation, identify geographic areas that may seek, but lack, access to the vaccines, and actively prepare for fluctuations in vaccination demands.

Exome sequencing of family trios from the National Birth Defects Prevention Study: Tapping into a rich resource of genetic and environmental data.

BACKGROUND: The National Birth Defects Prevention Study (NBDPS) is a multisite, population-based, case-control study of genetic and nongenetic risk factors for major structural birth defects. Eligible women had a pregnancy affected by a birth defect or a liveborn child without a birth defect between 1997 and 2011. They were invited to complete a telephone interview to collect pregnancy exposure data and were mailed buccal cell collection kits to collect specimens from themselves, their child (if living), and their child's father. Over 23,000 families representing more than 30 major structural birth defects provided DNA specimens. METHODS: To evaluate their utility for exome sequencing (ES), specimens from 20 children with colonic atresia were studied. Evaluations were conducted on specimens collected using cytobrushes stored and transported in open versus closed packaging, on native genomic DNA (gDNA) versus whole genome amplified (WGA) products and on a library preparation protocol adapted to low amounts of DNA. RESULTS: The DNA extracted from brushes in open packaging yielded higher quality sequence data than DNA from brushes in closed packaging. Quality metrics of sequenced gDNA were consistently higher than metrics from corresponding WGA products and were consistently high when using a low input protocol. CONCLUSIONS: This proof-of-principle study established conditions under which ES can be applied to NBDPS specimens. Successful sequencing of exomes from well-characterized NBDPS families indicated that this unique collection can be used to investigate the roles of genetic variation and gene-environment interaction effects in birth defect etiologies, providing a valuable resource for birth defect researchers.

Use of consumer survey data to target cessation messages to smokers through mass media.

OBJECTIVES: We identified the mass media channels that reach the most cigarette smokers in an attempt to more effectively target smoking cessation messages. METHODS: Reach estimates and index scores for smokers were taken from 2002-2003 ConsumerStyles and HealthStyles national surveys of adults (N=11660) to estimate overall and demographic-specific exposure measures for television, radio, newspapers, and magazines. RESULTS: Smokers viewed more television, listened to more radio, and read fewer magazines and newspapers than did nonsmokers. Nearly one third of smokers were regular daytime or late-night television viewers. Selected cable television networks (USA, Lifetime, and Discovery Channel) and selected radio genres, such as classic rock and country, had high reach and were cost-efficient channels for targeting smokers. CONCLUSIONS: Certain mass media channels offer efficient opportunities to target smoking cessation messages so they reach relatively large audiences of smokers at relatively low cost. The approach used in this study can be applied to other types of health risk factors to improve health communication planning and increase efficiency of program media expenditures.

The PhenoGen informatics website: tools for analyses of complex traits.

BACKGROUND: With the advent of "omics" (e.g. genomics, transcriptomics, proteomics and phenomics), studies can produce enormous amounts of data. Managing this diverse data and integrating with other biological data are major challenges for the bioinformatics community. Comprehensive new tools are needed to store, integrate and analyze the data efficiently. DESCRIPTION: The PhenoGen Informatics website http://phenogen.uchsc.edu is a comprehensive toolbox for storing, analyzing and integrating microarray data and related genotype and phenotype data. The site is particularly suited for combining QTL and microarray data to search for "candidate" genes contributing to complex traits. In addition, the site allows, if desired by the investigators, sharing of the data. Investigators can conduct "in-silico" microarray experiments using their own and/or "shared" data. CONCLUSION: The PhenoGen website provides access to tools that can be used for high-throughput data storage, analyses and interpretation of the results. Some of the advantages of the architecture of the website are that, in the future, the present set of tools can be adapted for the analyses of any type of high-throughput "omics" data, and that access to new tools, available in the public domain or developed at PhenoGen, can be easily provided.

Functional screen identifies regulators of murine hematopoietic stem cell repopulation.

Understanding the molecular regulation of hematopoietic stem and progenitor cell (HSPC) engraftment is paramount to improving transplant outcomes. To discover novel regulators of HSPC repopulation, we transplanted >1,300 mice with shRNA-transduced HSPCs within 24 h of isolation and transduction to focus on detecting genes regulating repopulation. We identified 17 regulators of HSPC repopulation: Arhgef5, Armcx1, Cadps2, Crispld1, Emcn, Foxa3, Fstl1, Glis2, Gprasp2, Gpr56, Myct1, Nbea, P2ry14, Smarca2, Sox4, Stat4, and Zfp251. Knockdown of each of these genes yielded a loss of function, except in the cases of Armcx1 and Gprasp2, whose loss enhanced hematopoietic stem cell (HSC) repopulation. The discovery of multiple genes regulating vesicular trafficking, cell surface receptor turnover, and secretion of extracellular matrix components suggests active cross talk between HSCs and the niche and that HSCs may actively condition the niche to promote engraftment. We validated that Foxa3 is required for HSC repopulating activity, as Foxa3(-/-) HSC fails to repopulate ablated hosts efficiently, implicating for the first time Foxa genes as regulators of HSPCs. We further show that Foxa3 likely regulates the HSC response to hematologic stress. Each gene discovered here offers a window into the novel processes that regulate stable HSPC engraftment into an ablated host.

Public policy to maximize tobacco cessation.

Tobacco use kills more than 400,000 Americans every year. For smokers, quitting is the biggest step they can take to improve their health, but it is a difficult step. Fortunately, policy-based interventions can both encourage smokers to quit and help them succeed. Evidence shows that tobacco tax increases encourage smokers to quit-recent state and federal increases have created dramatic surges in calls to quitlines. Similarly, smokefree workplace laws not only protect workers and patrons from secondhand smoke but also encourage smokers to quit, help them succeed, and create a social environment less conducive to smoking. The impact of policy changes can be amplified by promoting quitting around the date they are implemented. Outreach to health practitioners can alert them to encourage their patients to quit. Earned and paid media can also be used to motivate smokers to quit when policy changes are put into effect. Although these policies and efforts regarding them can generate great demand for evidence-based cessation services such as counseling and medication, it is important to make these resources available for those wanting to quit. Public and private health insurance plans should provide coverage for cessation services, and states should invest tobacco tax and/or tobacco settlement dollars in smoking-cessation programs as recommended by the CDC. Finally, the Family Smoking Prevention and Tobacco Control Act has given the U.S. Food and Drug Administration new authority to regulate tobacco products and marketing, and to prevent tobacco companies from deceptively marketing new products that discourage smokers from quitting and keep them addicted.