Sensory analysis of characterising flavours: evaluating tobacco product odours using an expert panel.

OBJECTIVES: Tobacco flavours are an important regulatory concept in several jurisdictions, for example in the USA, Canada and Europe. The European Tobacco Products Directive 2014/40/EU prohibits cigarettes and roll-your-own tobacco having a characterising flavour. This directive defines characterising flavour as 'a clearly noticeable smell or taste other than one of tobacco […]'. To distinguish between products with and without a characterising flavour, we trained an expert panel to identify characterising flavours by smelling. METHODS: An expert panel (n=18) evaluated the smell of 20 tobacco products using self-defined odour attributes, following Quantitative Descriptive Analysis. The panel was trained during 14 attribute training, consensus training and performance monitoring sessions. Products were assessed during six test sessions. Principal component analysis, hierarchical clustering (four and six clusters) and Hotelling's T-tests (95% and 99% CIs) were used to determine differences and similarities between tobacco products based on odour attributes. RESULTS: The final attribute list contained 13 odour descriptors. Panel performance was sufficient after 14 training sessions. Products marketed as unflavoured that formed a cluster were considered reference products. A four-cluster method distinguished cherry-flavoured, vanilla-flavoured and menthol-flavoured products from reference products. Six clusters subdivided reference products into tobacco leaves, roll-your-own and commercial products. CONCLUSIONS: An expert panel was successfully trained to assess characterising odours in cigarettes and roll-your-own tobacco. This method could be applied to other product types such as e-cigarettes. Regulatory decisions on the choice of reference products and significance level are needed which directly influences the products being assessed as having a characterising odour.

New Tobacco and Tobacco-Related Products: Early Detection of Product Development, Marketing Strategies, and Consumer Interest.

BACKGROUND: A wide variety of new tobacco and tobacco-related products have emerged on the market in recent years. OBJECTIVE: To understand their potential implications for public health and to guide tobacco control efforts, we have used an infoveillance approach to identify new tobacco and tobacco-related products. METHODS: Our search for tobacco(-related) products consists of several tailored search profiles using combinations of keywords such as "e-cigarette" and "new" to extract information from almost 9000 preselected sources such as websites of online shops, tobacco manufacturers, and news sites. RESULTS: Developments in e-cigarette design characteristics show a trend toward customization by possibilities to adjust temperature and airflow, and by the large variety of flavors of e-liquids. Additionally, more e-cigarettes are equipped with personalized accessories, such as mobile phones, applications, and Bluetooth. Waterpipe products follow the trend toward electronic vaping. Various heat-not-burn products were reintroduced to the market. CONCLUSIONS: Our search for tobacco(-related) products was specific and timely, though advances in product development require ongoing optimization of the search strategy. Our results show a trend toward products resembling tobacco cigarettes vaporizers that can be adapted to the consumers' needs. Our search for tobacco(-related) products could aid in the assessment of the likelihood of new products to gain market share, as a possible health risk or as an indicator for the need on independent and reliable information of the product to the general public.

Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation.

The Suv39h1 and Suv39h2 histone lysine methyltransferases are hallmark enzymes at mammalian heterochromatin. We show here that the mouse Suv39h2 enzyme differs from Suv39h1 by containing an N-terminal basic domain that facilitates retention at mitotic chromatin and provides an additional affinity for major satellite repeat RNA. To analyze an RNA-dependent interaction with chromatin, we purified native nucleosomes from mouse ES cells and detect that Suv39h1 and Suv39h2 exclusively associate with poly-nucleosomes. This association was attenuated upon RNaseH incubation and entirely lost upon RNaseA digestion of native chromatin. Major satellite repeat transcripts remain chromatin-associated and have a secondary structure that favors RNA:DNA hybrid formation. Together, these data reveal an RNA-mediated mechanism for the stable chromatin interaction of the Suv39h KMT and suggest a function for major satellite non-coding RNA in the organization of an RNA-nucleosome scaffold as the underlying structure of mouse heterochromatin.

A test strategy for the assessment of additive attributed toxicity of tobacco products.

The new EU Tobacco Product Directive (TPD) prohibits tobacco products containing additives that are toxic in unburnt form or that increase overall toxicity of the product. This paper proposes a strategy to assess additive attributed toxicity in the context of the TPD. Literature was searched on toxicity testing strategies for regulatory purposes from tobacco industry and governmental institutes. Although mainly traditional in vivo testing strategies have been applied to assess toxicity of unburnt additives and increases in overall toxicity of tobacco products due to additives, in vitro tests combined with toxicogenomics and validated using biomarkers of exposure and disease are most promising in this respect. As such, tests are needed that are sensitive enough to assess additive attributed toxicity above the overall toxicity of tobacco products, which can associate assay outcomes to human risk and exposure. In conclusion, new, sensitive in vitro assays are needed to conclude whether comparable testing allows for assessment of small changes in overall toxicity attributed to additives. A more pragmatic approach for implementation on a short-term is mandated lowering of toxic emission components. Combined with risk assessment, this approach allows assessment of effectiveness of harm reduction strategies, including banning or reducing of additives.

An inventory of methods suitable to assess additive-induced characterising flavours of tobacco products.

BACKGROUND: Products with strong non-tobacco flavours are popular among young people, and facilitate smoking initiation. Similar to the U.S. Food and Drug Administration Tobacco Control Act, the new European Tobacco Product Directive (TPD) prohibits cigarettes and roll-your-own tobacco with a characterising flavour other than tobacco. However, no methods are prescribed or operational to assess characterising flavours. This is the first study to identify, review and synthesize the existing peer-reviewed and tobacco industry literature in order to provide an inventory of methods suitable to assess characterising flavours. METHODS: Authors gathered key empirical and theoretical papers examining methods suitable to assess characterising flavours. Scientific literature databases (PubMed and Scopus) and tobacco industry documents were searched, based on several keyword combinations. Inclusion criteria were relevance for smoked tobacco products, and quality of data. RESULTS: The findings reveal that there is a wide variation in natural tobacco flavours. Flavour differences from natural tobacco can be described by both expert and consumer sensory panels. Most methods are based on smoking tests, but odour evaluation has also been reported. Chemical analysis can be used to identify and quantify levels of specific flavour additives in tobacco products. CONCLUSIONS: As flavour perception is subjective, and requires human assessment, sensory analysis in consumer or expert panel studies is necessitated. We recommend developing validated tests for descriptive sensory analysis in combination with chemical-analytical measurements. Testing a broad range of brands, including those with quite subtle characterizing flavours, will provide the concentration above which an additive will impart a characterising flavour.

An Inventory of Methods for the Assessment of Additive Increased Addictiveness of Tobacco Products.

BACKGROUND: Cigarettes and other forms of tobacco contain the addictive drug nicotine. Other components, either naturally occurring in tobacco or additives that are intentionally added during the manufacturing process, may add to the addictiveness of tobacco products. As such, these components can make cigarette smokers more easily and heavily dependent.Efforts to regulate tobacco product dependence are emerging globally. Additives that increase tobacco dependence will be prohibited under the new European Tobacco Product Directive. OBJECTIVE: This article provides guidelines and recommendations for developing a regulatory strategy for assessment of increase in tobacco dependence due to additives. Relevant scientific literature is summarized and criteria and experimental studies that can define increased dependence of tobacco products are described. CONCLUSIONS: Natural tobacco smoke is a very complex matrix of components, therefore analysis of the contribution of an additive or a combination of additives to the level of dependence on this product is challenging. We propose to combine different type of studies analyzing overall tobacco product dependence potential and the functioning of additives in relation to nicotine. By using a combination of techniques, changes associated with nicotine dependence such as behavioral, physiological, and neurochemical alterations can be examined to provide sufficient information.Research needs and knowledge gaps will be discussed and recommendations will be made to translate current knowledge into legislation. As such, this article aids in implementation of the Tobacco Product Directive, as well as help enable regulators and researchers worldwide to develop standards to reduce dependence on tobacco products. IMPLICATIONS: This article provides an overall view on how to assess tobacco product constituents for their potential contribution to use and dependence. It provides guidelines that help enable regulators worldwide to develop standards to reduce dependence on tobacco products and guide researches to set research priorities on this topic.

A transcription factor-based mechanism for mouse heterochromatin formation.

Heterochromatin is important for genome integrity and stabilization of gene-expression programs. We have identified the transcription factors Pax3 and Pax9 as redundant regulators of mouse heterochromatin, as they repress RNA output from major satellite repeats by associating with DNA within pericentric heterochromatin. Simultaneous depletion of Pax3 and Pax9 resulted in dramatic derepression of major satellite transcripts, persistent impairment of heterochromatic marks and defects in chromosome segregation. Genome-wide analyses of methylated histone H3 at Lys9 showed enrichment at intergenic major satellite repeats only when these sequences retained intact binding sites for Pax and other transcription factors. Additionally, bioinformatic interrogation of all histone methyltransferase Suv39h-dependent heterochromatic repeat regions in the mouse genome revealed a high concordance with the presence of transcription factor binding sites. These data define a general model in which reiterated arrangement of transcription factor binding sites within repeat sequences is an intrinsic mechanism of the formation of heterochromatin.

The male germ cell gene regulator CTCFL is functionally different from CTCF and binds CTCF-like consensus sites in a nucleosome composition-dependent manner.

BACKGROUND: CTCF is a highly conserved and essential zinc finger protein expressed in virtually all cell types. In conjunction with cohesin, it organizes chromatin into loops, thereby regulating gene expression and epigenetic events. The function of CTCFL or BORIS, the testis-specific paralog of CTCF, is less clear. RESULTS: Using immunohistochemistry on testis sections and fluorescence-based microscopy on intact live seminiferous tubules, we show that CTCFL is only transiently present during spermatogenesis, prior to the onset of meiosis, when the protein co-localizes in nuclei with ubiquitously expressed CTCF. CTCFL distribution overlaps completely with that of Stra8, a retinoic acid-inducible protein essential for the propagation of meiosis. We find that absence of CTCFL in mice causes sub-fertility because of a partially penetrant testicular atrophy. CTCFL deficiency affects the expression of a number of testis-specific genes, including Gal3st1 and Prss50. Combined, these data indicate that CTCFL has a unique role in spermatogenesis. Genome-wide RNA expression studies in ES cells expressing a V5- and GFP-tagged form of CTCFL show that genes that are downregulated in CTCFL-deficient testis are upregulated in ES cells. These data indicate that CTCFL is a male germ cell gene regulator. Furthermore, genome-wide DNA-binding analysis shows that CTCFL binds a consensus sequence that is very similar to that of CTCF. However, only ~3,700 out of the ~5,700 CTCFL- and ~31,000 CTCF-binding sites overlap. CTCFL binds promoters with loosely assembled nucleosomes, whereas CTCF favors consensus sites surrounded by phased nucleosomes. Finally, an ES cell-based rescue assay shows that CTCFL is functionally different from CTCF. CONCLUSIONS: Our data suggest that nucleosome composition specifies the genome-wide binding of CTCFL and CTCF. We propose that the transient expression of CTCFL in spermatogonia and preleptotene spermatocytes serves to occupy a subset of promoters and maintain the expression of male germ cell genes.

CTCF regulates the local epigenetic state of ribosomal DNA repeats.

BACKGROUND: CCCTC binding factor (CTCF) is a highly conserved zinc finger protein, which is involved in chromatin organization, local histone modifications, and RNA polymerase II-mediated gene transcription. CTCF may act by binding tightly to DNA and recruiting other proteins to mediate its various functions in the nucleus. To further explore the role of this essential factor, we used a mass spectrometry-based approach to screen for novel CTCF-interacting partners. RESULTS: Using biotinylated CTCF as bait, we identified upstream binding factor (UBF) and multiple other components of the RNA polymerase I complex as potential CTCF-interacting partners. Interestingly, CTCFL, the testis-specific paralog of CTCF, also binds UBF. The interaction between CTCF(L) and UBF is direct, and requires the zinc finger domain of CTCF(L) and the high mobility group (HMG)-box 1 and dimerization domain of UBF. Because UBF is involved in RNA polymerase I-mediated ribosomal (r)RNA transcription, we analyzed CTCF binding to the rDNA repeat. We found that CTCF bound to a site upstream of the rDNA spacer promoter and preferred non-methylated over methylated rDNA. DNA binding by CTCF in turn stimulated binding of UBF. Absence of CTCF in cultured cells resulted in decreased association of UBF with rDNA and in nucleolar fusion. Furthermore, lack of CTCF led to reduced binding of RNA polymerase I and variant histone H2A.Z near the rDNA spacer promoter, a loss of specific histone modifications, and diminished transcription of non-coding RNA from the spacer promoter. CONCLUSIONS: UBF is the first common interaction partner of CTCF and CTCFL, suggesting a role for these proteins in chromatin organization of the rDNA repeats. We propose that CTCF affects RNA polymerase I-mediated events globally by controlling nucleolar number, and locally by regulating chromatin at the rDNA spacer promoter, similar to RNA polymerase II promoters. CTCF may load UBF onto rDNA, thereby forming part of a network that maintains rDNA genes poised for transcription.

Critical role for the transcription regulator CCCTC-binding factor in the control of Th2 cytokine expression.

Differentiation of naive CD4+ cells into Th2 cells is accompanied by chromatin remodeling at the Th2 cytokine locus allowing the expression of the IL-4, IL-5, and IL-13 genes. In this report, we investigated the role in Th2 differentiation of the transcription regulator CCCTC-binding factor (CTCF). Chromatin immunoprecipitation analysis revealed multiple CTCF binding sites in the Th2 cytokine locus. Conditional deletion of the Ctcf gene in double-positive thymocytes allowed development of peripheral T cells, but their activation and proliferation upon anti-CD3/anti-CD28 stimulation in vitro was severely impaired. Nevertheless, when TCR signaling was circumvented with phorbol ester and ionomycin, we observed proliferation of CTCF-deficient T cells, enabling the analysis of Th2 differentiation in vitro. We found that in CTCF-deficient Th2 polarization cultures, transcription of IL-4, IL-5, and IL-13 was strongly reduced. By contrast, CTCF deficiency had a moderate effect on IFN-gamma production in Th1 cultures and IL-17 production in Th17 cultures was unaffected. Consistent with a Th2 cytokine defect, CTCF-deficient mice had very low levels of IgG1 and IgE in their serum, but IgG2c was close to normal. In CTCF-deficient Th2 cultures, cells were polarized toward the Th2 lineage, as substantiated by induction of the key transcriptional regulators GATA3 and special AT-rich binding protein 1 (SATB1) and down-regulation of T-bet. Also, STAT4 expression was low, indicating that in the absence of CTCF, GATA3 still operated as a negative regulator of STAT4. Taken together, these findings show that CTCF is essential for GATA3- and SATB1-dependent regulation of Th2 cytokine gene expression.

CTCF regulates cell cycle progression of alphabeta T cells in the thymus.

The 11-zinc finger protein CCCTC-binding factor (CTCF) is a highly conserved protein, involved in imprinting, long-range chromatin interactions and transcription. To investigate its function in vivo, we generated mice with a conditional Ctcf knockout allele. Consistent with a previous report, we find that ubiquitous ablation of the Ctcf gene results in early embryonic lethality. Tissue-specific inactivation of CTCF in thymocytes specifically hampers the differentiation of alphabeta T cells and causes accumulation of late double-negative and immature single-positive cells in the thymus of mice. These cells are normally large and actively cycling, and contain elevated amounts of CTCF. In Ctcf knockout animals, however, these cells are small and blocked in the cell cycle due to increased expression of the cyclin-CDK inhibitors p21 and p27. Taken together, our results show that CTCF is required in a dose-dependent manner and is involved in cell cycle progression of alphabeta T cells in the thymus. We propose that CTCF positively regulates cell growth in rapidly dividing thymocytes so that appropriate number of cells are generated before positive and negative selection in the thymus.