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This research studied anti-gnawing bio-polymer composites to prevent rodent destroy the plastic products. Poly(butylene succinate) (PBS) used as a bio-polymer matrix incorporated with capsicum oleoresin (CO) and denatonium benzoate (DB). CO extracted form hot chili pepper provided the burning sensation to mammal and DB gave a bitterest taste as a non-toxic substance. CO liquid was impregnated in the porous of silica called SiCO. The anti-gnawing bio-polymer composites were prepared by internal mixer and formed the specimens by compression molding machine. The anti-gnawing bio-polymer composites were determined anti-gnawing properties and mechanical properties. The anti-gnawing properties was examined in term of percentage of weight loss monitoring every week for one month. The results showed that the bio-polymer composites could be able to withstand the gnawing from rats with decreasing percentage of weight loss around 16% as comparing to neat PBS; however, the increase in SiCO concentration did not significantly improve the anti-gnawing properties. The addition of SiCO did not significantly reduce mechanical properties of the bio-polymer composites; however the corporation of DB/ZnSt decreased those properties of the composites.

Heterogeneous tissue models require the assembly and co-culture of multiple types of cells. Our recent work demonstrated taste signal transmission from gustatory cells to neurons by grafting single-stranded DNA into the cell membrane to construct multicellular assemblies. However, the weak DNA linkage and low grafting density allowed the formation of large gustatory cell self-aggregates that cannot communicate with neurons efficiently. This article presents the construction of artificial taste buds exhibiting active intercellular taste signal transmission through the hybridization of gustatory-neuronal multicellular interfaces using bioorthogonal click chemistry. Hybrid cell clusters were formed by the self-assembly of neonatal gustatory cells displaying tetrazine with a precultured embryonic hippocampal neuronal network displaying trans-cyclooctene. A bitter taste signal transduction was provoked in gustatory cells using denatonium benzoate and transmitted to neurons as monitored by intracellular calcium ion sensing. In the multicellular hybrids, the average number of signal transmissions was five to six peaks per cell, and the signal transmission lasted for ∼5 min with a signal-to-signal gap time of 10-40 s. The frequent and extended intercellular signal transmission suggests that the cell surface modification by the bioorthogonal click chemistry is a promising approach to fabricating functional multicellular hybrid clusters potentially useful for cell-based biosensors, toxicity assays, and tissue regeneration.

Bitter taste receptors (T2Rs) are G protein-coupled receptors (GPCRs) expressed in various cell types including ciliated airway epithelial cells and macrophages. T2Rs in these two airway innate immune cell types are activated by bitter products, including some secreted by Pseudomonas aeruginosa, leading to Ca2+-dependent activation of endothelial nitric oxide (NO) synthase (eNOS). NO enhances mucociliary clearance and has direct antibacterial effects in ciliated epithelial cells and increases phagocytosis by macrophages. Using biochemistry and live cell imaging, we explored the role of heat shock protein 90 (HSP90) in regulating T2R-dependent NO pathways in primary sinonasal epithelial cells, primary monocyte-derived macrophages, and a human bronchiolar cell line (H441). We used immunofluorescence to show that H441 cells express eNOS and certain T2Rs and that the bitterant denatonium benzoate activates NO production in an HSP90-dependent manner in cells grown either as submerged cultures and at air liquid interface. In primary sinonasal epithelial cells, we determined that HSP-90 inhibition reduces T2R-stimulated NO production and ciliary beating which are crucial for pathogen clearance. In primary monocyte-derived macrophages, we found that HSP-90 is integral to T2R-stimulated NO production and phagocytosis of FITC-labeled Escherichia coli and pHrodo-Staphylococcus aureus. Our study demonstrates that HSP90 serves an innate immune role by regulating NO production downstream of T2R signaling by augmenting eNOS activation without impairing upstream calcium signaling. These findings suggest that HSP90 plays an important role in airway antibacterial innate immunity and may be an important target in airway diseases like chronic rhinosinusitis, asthma, or cystic fibrosis.

Purpose: To evaluate the effect of denatonium benzoate (DB) in histamine-induced model of inflammation and the effect of the selective H1 receptor agonist (2-(2-Pyridyl) ethylamine) on rat gastric smooth muscle strips pretreated with DB.Methods: The anti-inflammatory effect of DB was evaluated in vivo on histamine-induced rat paw edema. In vitro studies on spontaneous muscle contraction were performed on smooth muscle strips isolated from rat gastric corpus.Results: The results showed a well-defined anti-inflammatory effect of DB (15 mg/kg) during the early stage of rat paw edema at the 15th (p < 0.001), 30th (p < 0.01) and 60th min (p < 0.001) compared to control. In vitro experiments indicated reduced spontaneous contractile activity of smooth muscle strips to H1 receptor agonist in the presence of DB (0.5 μM). The vascular effects of histamine are mediated by H1 receptors. Substances, which reduce the effect of histamine on the H1 receptors could influence the early stage of histamine-induced inflammation.Conclusion: The results show that the anti-inflammatory activity of DB probably is related to its antagonistic activity on histamine H1 receptors. The results would contribute to the search for new antiinflammatory drugs. Keywords: Denatonium benzoate, Inflammation, Histamine, Muscle contraction

Bitter taste substances commonly represent a signal of toxicity. Fast and reliable detection of bitter molecules improves the safety of foods and beverages. Here, we report a biosensor using an easily accessible and cost-effective odorant-binding protein (OBP) of Drosophila melanogaster as a biosensitive material for the detection of bitter molecules. Based on the theoretical evaluation of the protein–ligand interaction, binding energies between the OBP and bitter molecules were calculated via molecular docking for the prediction and verification of binding affinities. Through one-step reduction, gold nanoparticles (AuNPs) and reduced graphene oxide (rGO) were deposited on the screen-printed electrodes for improving the electrochemical properties of electrodes. After the electrodes were immobilized with OBPs via layer-by-layer self-assembly, typical bitter molecules, such as denatonium, quinine, and berberine, were investigated through electrochemical impedance spectroscopy. The bitter molecules showed significant binding properties to the OBP with linear response concentrations ranging from 10–9 to 10–6 mg/mL. Therefore, the OBP-based biosensor offered powerful analytic techniques for the detection of bitter molecules and showed promising applications in the field of bitter taste evaluation.

Bitter taste receptors (taste 2 receptors, TAS2Rs) serve as warning sensors in the lingual system against the ingestion of potentially poisonous food. Here, we investigated the functional role of TAS2Rs in the human gut and focused on their potential to trigger an additional host defense pathway in the intestine. Human jejunal crypts, especially those from individuals with obesity, responded to bitter agonists by inducing the release of antimicrobial peptides (α-defensin 5 and regenerating islet-derived protein 3 α [REG3A]) but also regulated the expression of other innate immune factors (mucins, chemokines) that affected E. coli growth. We found that the effect of aloin on E. coli growth and on the release of the mucus glycoprotein CLCA1, identified via proteomics, was affected by TAS2R43 deletion polymorphisms and thus confirmed a role for TAS2R43. RNA-Seq revealed that denatonium benzoate induced an NRF2-mediated nutrient stress response and an unfolded protein response that increased the expression of the mitokine GDF15 but also ADM2 and LDLR, genes that are involved in anorectic signaling and lipid homeostasis. In conclusion, TAS2Rs in the intestine constitute a promising target for treating diseases that involve disturbances in the innate immune system and body weight control. TAS2R polymorphisms may be valuable genetic markers to predict therapeutic responses. ispartof: JOURNAL OF CLINICAL INVESTIGATION vol:132 issue:3 ispartof: location:United States status: published

Background: Denatonium benzoate (DB), one of the bitterest compounds known to man, is currently added to a wide range of products and is also used for alcohol denaturation. Some reports demonstrated that asthmatic symptoms are associated with DB exposure but the possible links between DB and IgE-mediated allergy susceptibility have not been examined to date. We investigated the effects of DB on IgE-mediated mast cell degranulation in vitro and in the ovalbumin (OVA)-induced mouse model of allergy.Methods: DB treatments were given to RBL-2H3 IgE-sensitized rat mast cell/basophil cells and KU812 human basophilic cells together with OVA-induced allergic BALB/c mice. Allergic mediator release, Ca2+ influx and OVA-specific IgE anaphylactic shock symptoms were measured along with the cell-surface expression of the α-subunit of high-affinity IgE receptor FcεRI on mast cells.Results: DB increases β-hexosaminidase (β-hex) release and Ca2+ mobilization in IgE-mediated activated RBL-2H3 and KU812 cells, and enhanced the cell-surface expression of FcεRIα. DB also promoted the severity of OVA-induced anaphylactic and diarrheic symptoms which was accompanied by mucus thickness in jejunum and the levels of β-hex, histamine and OVA-specific IgE in allergy mice, as well as the levels of FcεRIα mRNA and the FcεRIα proteinin isolated mucosal mast cells. Conclusions: DB treatments can promote the IgE-mediated mast cell degranulation in vitro and OVA-induced allergic susceptibility in mice by upregulating mast-cell-surface FcεR1α expression, providing evidence for DB exposure in promoting allergy susceptibility.

Current understanding of functional characteristics and biochemical pathways in taste bud cells have been hindered due the lack of long-term cultured cells. To address this, we developed a holistic approach to fully characterise long term cultured bovine taste bud cells (BTBCs). Initially, cultured BTBCs were characterised using RT-PCR gene expression profiling, immunocytochemistry, flowcytometry and calcium imaging, that confirmed the cells were mature TBCs that express taste receptor genes, taste specific protein markers and capable of responding to taste stimuli, i.e., denatonium (2 mM) and quinine (462.30 μM). Gene expression analysis of forty-two genes implicated in taste transduction pathway (map04742) using custom-made RT-qPCR array revealed high and low expressed genes in BTBCs. Preliminary datamining and bioinformatics demonstrated that the bovine α-gustducin, gustatory G-protein, have higher sequence similarity to the human orthologue compared to rodents. Therefore, results from this work will replace animal experimentation and provide surrogate cell-based throughput system to study human taste transduction.

Oral vaccination of dogs against rabies has the potential to achieve mass coverage and thus deplete the virus of its most important reservoir host species. There is, however, no established non-invasive method to evaluate vaccine release in the oral cavity, following bait ingestion. In this study, two pre-selected marker methods in conjunction with their acceptance were assessed in local Thai dogs. Shelter dogs (n = 47) were offered one of four randomized bait formulations, bait type A-, containing Green S (E142) in a fructose solution, type B-, containing Patent Blue V (E131) in a fructose solution, type C-, containing the medium used for delivery of oral rabies vaccine in baits commercially produced, and type D-, containing denatonium benzoate, which was to serve as the negative control, due to its perceived bitterness. Patent Blue V was found to possess overall stronger dyeing capacities compared to Green S. Furthermore, there was no significant difference in the acceptance or bait handling of Patent Blue V baits compared to those containing the oral rabies vaccine medium alone, suggesting the potential use of this dye as a surrogate for rabies vaccine when testing newly developed bait formats.

In mammalian taste buds, Type I cells comprise half of all cells. These are termed “glial-like” based on morphologic and molecular features, but there are limited studies describing their function. We tested whether Type I cells sense chemosensory activation of adjacent chemosensory (i.e., Types II and III) taste bud cells, similar to synaptic glia. UsingGad2;;GCaMP3 mice of both sexes, we confirmed by immunostaining that, within taste buds, GCaMP expression is predominantly in Type I cells (with no Type II and ≈28% Type III cells expressing weakly). In dissociated taste buds, GCaMP+ Type I cells responded to bath-applied ATP (10-100 μm) but not to 5-HT (transmitters released by Type II or III cells, respectively). Type I cells also did not respond to taste stimuli (5 μmcycloheximide, 1 mmdenatonium). In lingual slice preparations also, Type I cells responded to bath-applied ATP (10-100 μm). However, when taste buds in the slice were stimulated with bitter tastants (cycloheximide, denatonium, quinine), Type I cells responded robustly. Taste-evoked responses of Type I cells in the slice preparation were significantly reduced by desensitizing purinoceptors or by purinoceptor antagonists (suramin, PPADS), and were essentially eliminated by blocking synaptic ATP release (carbenoxolone) or degrading extracellular ATP (apyrase). Thus, taste-evoked release of afferent ATP from type II chemosensory cells, in addition to exciting gustatory afferent fibers, also activates glial-like Type I taste cells. We speculate that Type I cells sense chemosensory activation and that they participate in synaptic signaling, similarly to glial cells at CNS tripartite synapses.SIGNIFICANCE STATEMENTMost studies of taste buds view the chemosensitive excitable cells that express taste receptors as the sole mediators of taste detection and transmission to the CNS. Type I “glial-like” cells, with their ensheathing morphology, are mostly viewed as responsible for clearing neurotransmitters and as the “glue” holding the taste bud together. In the present study, we demonstrate that, when intact taste buds respond to their natural stimuli, Type I cells sense the activation of the chemosensory cells by detecting the afferent transmitter. Because Type I cells synthesize GABA, a known gliotransmitter, and cognate receptors are present on both presynaptic and postsynaptic elements, Type I cells may participate in GABAergic synaptic transmission in the manner of astrocytes at tripartite synapses.

Bitter taste receptors (TAS2R) are expressed in many non-sensor tissues including skeletal muscles but their function remains unexplored. The aim of this study is to investigate the role of TAS2R in rat abdominal skeletal muscles contractions using denatonium, a TAS2R agonist. Low concentration of denatonium (0.01 mmol/l) caused a significant decrease of amplitudes of the electrical field stimulation (EFS)-induced contractions in abdominal skeletal muscles preparations in vitro. This inhibitory effect was significantly reduced when the preparations were pre-incubated with gentamicin (0.02 mmol/l) used as a non-specific inhibitor of IP3 formation or with BaCl(2) (0.03 mmol/l) applied to block the inward-rectifier potassium current. All experiments were performed in the presence of pipecuronium in order to block the nerve stimulation of the contractions. The data obtained suggest that denatonium decreases the force of rat abdominal muscles contractions mainly via activation of TAS2R, phosphatidylinositol 4,5-biphosphate and its downstream signal metabolites.

Denatonium, one of the bitterest substances known to man, was recently identified as wastewater borne micropollutant in surface waters. Therefore, photodegradation experiments and electrochemical degradation were performed to identify abiotic and putative biotic transformation products (TPs). Indirect rather than direct photodegradation proved to be important for denatonium removal by solar irradiation and produced seven TPs. Amide hydrolysis, hydroxylation, N-dealkylation, and N-dearylation were revealed as the main mechanisms. Anodic oxidation of denatonium was related to the formation of overall ten products and despite considerable different yields, all TPs from indirect photodegradation were mimicked electrochemically. Among them, lidocaine was the only TP detected after conventional wastewater treatment and in surface waters. The occurrence of lidocaine was however associated with its application as local anesthetic rather than to a degradation of denatonium. The absence of additional products suggests that denatonium degradation is negligible under environmental conditions, supporting the previously described persistent nature of this compound. Advanced water treatment techniques however have the potential to degrade denatonium. About 74% of the initial denatonium load was removed from wastewater during pilot-scale ozonation. The degradation of denatonium was accompanied here with the formation of at least two polar products, which are passing unchanged through a sand filter after ozonation. Both substances have completely unknown (toxicological) properties and this study seems to be the first report about their structures in general, as none of them was found in any of the large compound libraries (e.g. PubChem).

Taste stimulants play important roles in triggering digestion and absorption of nutrients and in toxin detection, under the control of the gut-brain axis. Bitter compounds regulate gut hormone secretion and gastrointestinal motility through bitter taste receptors (TAS2Rs) located in the taste buds on the tongue and in the enteroendocrine cells. Gastric accommodation (GA) is an important physiologic function. However, the role of TAS2R agonists in regulating GA remains unclear. To clarify whether GA is influenced by bitter stimulants, we examined the effect of TAS2R agonist denatonium benzoate (DB), administered intraorally and intragastrically, by measuring the consequent intrabag pressure in the proximal stomach of guinea pigs. Effects of the Kampo medicine rikkunshito (RKT) and its bitter components liquiritigenin and naringenin on GA were also examined. Intraoral DB (0.2 nmol/ml) administration enhanced GA. Intragastric DB administration (0.1 and 1 nmol/kg) promoted GA, whereas higher DB doses (30 μmol/kg) inhibited it. Similar changes in GA were observed with intragastric (1000 mg/kg) and intraoral (200 mg/ml) RKT administration. Liquiritigenin and naringenin also promoted GA. These findings suggest that GA is affected by the stimulation of TAS2Rs in the oral cavity or gut in guinea pigs.

Chemosensory signaling in organs such as the mouth and gut contributes to the mechanisms that control metabolism. We investigated the chemosensory pathways that regulate secretion of the hunger hormone ghrelin in response to neurotransmitters, bitter and sweet tastants at the cellular level in the human gut mucosa, and the disturbances in this regulatory pathway induced by obesity. Obesity impaired ghrelin protein production and adrenalin-induced ghrelin secretion in fundic cells, which was counterbalanced by somatostatin. Bitter agonists selective for taste receptor type 2 (TAS2Rs), TAS2R5 and TAS2R10 stimulated ghrelin secretion in fundic cells. The stimulatory effect of the broadly tuned bitter agonist, denatonium benzoate, was selectively blunted by obesity in the small intestine but not in the fundus. Luminal glucose concentrations inhibited ghrelin secretion via sodium-dependent glucose cotransporter and taste receptor type 1 member 3. Obesity altered the sensitivity of the ghrelin cell to glucose in the small intestine but not in the fundus. Sweet taste receptor activation inhibited bitter taste signaling of the ghrelin cell. In conclusion, obesity impairs the sympathetic drive that controls ghrelin release in the fundus and affects the sensitivity of the ghrelin cell to bitter and sweet stimuli in the small intestine but not in the fundus. Region-selective targeting of gut taste receptors in obesity is indicated.-Wang, Q., Liszt, K. I., Deloose, E., Canovai, E., Thijs, T., Farré, R., Ceulemans, L. J., Lannoo, M., Tack, J., Depoortere, I. Obesity alters adrenergic and chemosensory signaling pathways that regulate ghrelin secretion in the human gut. ispartof: FASEB JOURNAL vol:33 issue:4 pages:4907-4920 ispartof: location:United States status: published

Denatonium benzoate (DB), a commonly used bitter agent in numerous products, has recently been recognized as a waterborne contaminant due to concern about its potential persistence, mobility and toxicity (PMT). However, its occurrence, levels and fate in global aquatic environments are largely unknown. In this study, a new sampling method, based on diffusive gradients in thin films (DGT) with mixed-mode cation exchange (MCX) as the binding agent, was developed for measuring DB in waters. MCX shows a rapid adsorption and high capacity for DB. DB is linearly accumulated by MCX-DGT. pH (6–8), ionic strength (0.01–0.5 M), or DOM (0–10 M) do not show any significant effect on the MCX-DGT performance, confirming its reliability. The DGT measurements in a wastewater treatment plant (WWTP) are comparable to those by paralleled grab sampling. The field results suggest DB is persistent in WWTPs and could be a potential domestic wastewater indicator. Therefore, MCX-DGT is a promising technique for understanding the environmental occurrence, levels and fate of DB. This is a first report of using DGT for DB monitoring and of DB occurrence in Chinese environments. Further exploration of DGT as a reliable passive monitoring tool for a wide range of PMT substances in different applications is warranted.

BACKGROUND Administration of a bitter compound can alter the intragastric pressure (IGP) after a meal. Additionally, a negative correlation between IGP and the number of transient lower esophageal sphincter relaxations (TLESRs) has been demonstrated. However, the effect of a bitter tastant on the number of TLESRs and subsequent reflux episodes has never been investigated and it is unclear whether bitter food items should be avoided in gastro-esophageal reflux disease. We hypothesize that bitter administration in healthy volunteers (HVs) will lead to an increase in the number of TLESRs. METHODS After an overnight fast, 20 female HVs (36 years [21-63]) underwent a high-resolution impedance manometry (HRiM) measurement. After placement of the HRiM probe, 0.1 ml/kg of a 10 mM denatonium benzoate solution (bitter) or an identical volume of water (placebo) was administered directly into the stomach. The number of TLESRs and reflux episodes was quantified 30 min before and 2 h after consumption of a high caloric meal. KEY RESULTS There was no significant difference in the number of TLESRs or reflux episodes between the bitter and placebo condition. Additionally, no differences were observed in the nature (gas or liquid) and extent of reflux events. Lower esophageal sphincter pressures dropped significantly in the first postprandial hour to start recovering slowly back to baseline values during the second postprandial hour (p

BACKGROUND: Bitter and sweet taste receptors are present in the human upper airway, where they have roles in innate immunity. Previous studies have shown that one of the 25 bitter receptors, TAS2R38, responds to specific bacterial signaling molecules and evokes one type of a defense response in the upper airway, whereas ligands of sweet receptors suppress other types of defense responses. METHODS: We examined whether other bitter taste receptors might also be involved in innate immunity by using sensory responses to bitter compounds that are not ligands of TAS2R38 (quinine and denatonium benzoate) to assess the sensitivity of other bitter receptors in chronic rhinosinusitis (CRS) patients. CRS patients with (N=426) and without (N=226) nasal polyps and controls (N=356) rated the intensity of quinine, denatonium benzoate, phenylthiocarbamide (PTC; a ligand for TAS2R38), sucrose, and salt. RESULTS: CRS patients rated the bitter compounds denatonium benzoate and quinine as less intense and sucrose as more intense than did controls (FDR0.05). PTC bitter taste intensity differed between patient and control groups but were less marked than those previously reported. Though differences were statistically significant, overall effect sizes were small. CONCLUSION: CRS patients report bitter stimuli as less intense but sweet stimuli as more intense than do control subjects. We speculate that taste responses may reflect the competence of sinonasal innate immunity mediated by taste receptor function, and thus a taste test may have potential for clinical utility in CRS patients.

BACKGROUND Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP), and aspirin-exacerbated respiratory disease (AERD) have varying levels of inflammation and disease severity. Solitary chemosensory cells (SCCs) are enriched in nasal polyps, are the primary source of interleukin 25 (IL-25) in upper airways, leading to type 2 inflammation, and are activated by bitter-tasting denatonium benzoate (DB). Thus, we sought to evaluate DB taste perception at a range of concentrations in order to identify 1 that most differentiates CRS subgroups from controls. METHODS CRSsNP (n = 25), CRSwNP (n = 26), and AERD (n = 27) patients as well as controls (n = 25) tasted 6 DB concentrations in a fixed, random order, rating on a category scale of 0 (no intensity) to 12 (extremely intense). Sinonasal epithelial cultures were treated with and without denatonium and analyzed for IL-25 via flow cytometry. RESULTS CRSsNP patients rated DB as significantly less intense than did controls at all concentrations: 5.62 × 10-9 M, 1.00 × 10-8 M, 1.78 × 10-8 M, 3.16 × 10-8 M, 5.62 × 10-8 M, and 1.00 × 10-7 M (all p < 0.0083). CRSwNP patients did not show significant differences from controls. AERD patients rated DB as significantly more intense than did controls at concentrations of 1.00 × 10-8 M and 3.16 × 10-8 M (p < 0.0083). In vitro data demonstrated significant increase in IL-25-positive cells after denatonium stimulation (n = 5), compared to control (n = 5) (p = 0.012). CONCLUSION Our findings link in vitro DB stimulation of sinonasal tissue with increased IL-25 and show differential DB taste perception in CRS subgroups relative to the control group, with CRSsNP being hyposensitive and AERD being hypersensitive. We propose a concentration of 3.16 × 10-8 M for future study of clinical utility.

Background Bitter tastants can activate bitter taste receptors (TAS2Rs) and thus initiate relaxation of airway smooth muscle cells (ASMCs), which have great potential in the development of novel bronchodilator drugs for asthma therapy. However, the canonical bitter substance, denatonium is known to induce apoptosis of airway epithelial cells (AECs), indicating that other bitter tastants may also impair the epithelial integrity to prevent hazardous particulate matters such as coronaviruses. Therefore, any bitter tastants intended for treating airway disease should be carefully evaluated for potential toxicity to AECs. Hypothesis/Purpose Considering the vast diversity of bitter tastants in nature and different types of TAS2Rs expressed in airway cells, we hypothesized that there must be some natural bitter tastants to be not only potent in inducing relaxation of ASMCs but also unharmful to AECs. Study design and methods Here we evaluated a group of bitter flavonoids that are derived from fruits and commonly used in traditional herbal medicine, including apigenin, hesperetin, kaempferol, naringenin, quercetin, and naringin, for their effects on the proliferation of human airway epithelial-like (16HBE14o-, BEAS-2B, and A549) cells cultured in vitro. Cell proliferation and associated signaling pathways were assessed by cell counting, ATP assay, cell cycling assay, quantitative RT-PCR, Fluo-4 labeling, and fluorescence resonance energy transfer, respectively. Results The results show that five of the six tested bitter tastants inhibited, but only naringin promoted the proliferation of the 16HBE14o-, BEAS-2B, and A549 cells at the dose of a few hundred micromoles. Furthermore, the naringin-promoted proliferation of the 16HBE14o- cells was associated with enhanced cell cycle progression, mRNA expression of cyclin E, and evoked calcium signaling/ERK signaling, which were all attenuated by inhibition of the TAS2R signaling pathways with specific blockers. Conclusion These findings indicate that although the majority of the bitter flavonoids may inhibit the proliferation of AECs, naringin emerged as one to promote the proliferation of AECs via cell cycle progression and TAS2R-activated intracellular signaling. It suggests that naringin and not a few other bitter tastants can be proven with nontoxicity to the airway epithelial structure and function, which provides further confidence in the development of safe and effective TAS2R-based bronchodilators for asthma therapy.

For protection from inhaled pathogens many strategies have evolved in the airways such as mucociliary clearance and cough. We have previously shown that protective respiratory reflexes to locally released bacterial bitter “taste” substances are most probably initiated by tracheal brush cells (BC). Our single‐cell RNA‐seq analysis of murine BC revealed high expression levels of cholinergic and bitter taste signaling transcripts (Tas2r108, Gnat3, Trpm5). We directly demonstrate the secretion of acetylcholine (ACh) from BC upon stimulation with the Tas2R agonist denatonium. Inhibition of the taste transduction cascade abolished the increase in [Ca\(^{2+}\)]\(_{i}\) in BC and subsequent ACh‐release. ACh‐release is regulated in an autocrine manner. While the muscarinic ACh‐receptors M3R and M1R are activating, M2R is inhibitory. Paracrine effects of ACh released in response to denatonium included increased [Ca\(^{2+}\)]\(_{i}\) in ciliated cells. Stimulation by denatonium or with Pseudomonas quinolone signaling molecules led to an increase in mucociliary clearance in explanted tracheae that was Trpm5‐ and M3R‐mediated. We show that ACh‐release from BC via the bitter taste cascade leads to immediate paracrine protective responses that can be boosted in an autocrine manner. This mechanism represents the initial step for the activation of innate immune responses against pathogens in the airways.

Human perception of bitter substances is partially genetically determined. Previously we discovered a single nucleotide polymorphism (SNP) within the bitter taste receptor gene TAS2R19 on chromosome 12 that accounts for 5.8% of the variance in the perceived intensity rating of quinine, and we strengthened the classic association between TAS2R38 genotype and the bitterness of propylthiouracil (PROP). Here we performed a genome-wide association study (GWAS) using a 40% larger sample (n = 1999) together with a bivariate approach to detect previously unidentified common variants with small effects on bitter perception. We identified two signals, both with small effects (< 2%), within the bitter taste receptor clusters on chromosomes 7 and 12, which influence the perceived bitterness of denatonium benzoate and sucrose octaacetate respectively. We also provided the first independent replication for an association of caffeine bitterness on chromosome 12. Furthermore, we provided evidence for pleiotropic effects on quinine, caffeine, sucrose octaacetate and denatonium benzoate for the three SNPs on chromosome 12 and the functional importance of the SNPs for denatonium benzoate bitterness. These findings provide new insights into the genetic architecture of bitter taste and offer a useful starting point for determining the biological pathways linking perception of bitter substances.

The sense of bitter taste is critical for chickens to acquire and select feeds. It is important to understand the roles and mechanisms of bitter taste transduction in chickens. Denatonium is extensively used as a bitter taste receptor agonist to activate bitter taste receptors in recent studies. The objective of this study was to investigate the physiological effects and the potential molecular mechanisms of dietary exposure to a strong bitter taste receptor agonist on the jejunal epithelial cells of yellow-feathered chickens. A total of 240 yellow-feathered chickens were divided into four treatments receiving a normal diet (Control), a low-dose denatonium treatment (Control + 5 mg/kg denatonium), a middle-dose denatonium treatment (Control + 20 mg/kg denatonium) and a high-dose denatonium treatment (Control + 100 mg/kg denatonium) for 56 days, respectively. The results showed that dietary denatonium reduced (P < 0.05) the growth performance of chickens. High-dose denatonium damaged the morphology of the jejunal epithelium and decreased (P < 0.05) the activities of Ca2+-ATPase, sucrase and maltase after 56 days of exposure. Meanwhile, high-dose denatonium increased (P < 0.05) mRNA expressions of bitter taste receptors, which resulted in enhanced apoptosis in jejunal epithelial cells after 56 days of exposure. Furthermore, middle-dose and high-dose denatonium exhibited increased (P < 0.05) mRNA level of claudin 2 and decreased (P < 0.05) mRNA level of occludin after 28 days of exposure. Only high-dose denatonium decreased (P < 0.05) mRNA level of occludin after 56 days of exposure. In conclusion, denatonium manifested deleterious effects on the jejunum of chickens in a dose–effect manner via damaging the morphology of the jejunal epithelium, and inducing apoptosis associated with bitter taste receptors. Our data suggest that bitter-tasting feed additives may have side effects on the growth and development of intestines in chickens.

Animals must detect aversive compounds to survive. Bitter taste neurons express heterogeneous combinations of bitter receptors that diversify their response profiles, but this remains poorly understood. Here we describe groups of taste neurons in Drosophila that detect the same bitter compounds using unique combinations of gustatory receptors (GRs). These distinct complexes also confer responsiveness to non-overlapping sets of additional compounds. While either GR32a/GR59c/GR66a or GR22e/GR32a/GR66a heteromultimers are sufficient for lobeline, berberine, and denatonium detection, only GR22e/GR32a/GR66a responds to strychnine. Thus, despite minimal sequence-similarity, Gr22e and Gr59c show considerable but incomplete functional overlap. Since the gain- or loss-of-function of Gr22e or Gr59c alters bitter taste response profiles, we conclude a taste neuron’s specific combination of Grs determines its response profile. We suspect the heterogeneity of Gr expression in Drosophila taste neurons diversifies bitter compound detection, improving animal fitness under changing environmental conditions that present a variety of aversive compounds., Taste sensilla are Drosophila sensory organs containing taste neurons, which have differential tuning for bitter compounds. Here, the authors systematically examine what combinations of gustatory receptor genes confer a specific taste response profile in different bitter taste neurons.

Background Sinonasal bitter taste receptors (T2Rs) contribute to upper airway innate immunity and correlate with chronic rhinosinusitis (CRS) clinical outcomes. A subset of T2Rs expressed on sinonasal solitary chemosensory cells (SCCs) are activated by denatonium, resulting in a calcium-mediated secretion of bactericidal antimicrobial peptides (AMPs) in neighboring ciliated epithelial cells. We hypothesized that there is patient variability in the amount of bacterial killing induced by different concentrations of denatonium and that the differences correlate with CRS clinical outcomes. Methods Bacterial growth inhibition was quantified after mixing bacteria with airway surface liquid (ASL) collected from denatonium-stimulated sinonasal air-liquid interface (ALI) cultures. Patient ASL bacterial killing at 0.1 mM denatonium and baseline characteristics and sinus surgery outcomes were compared between these populations. Results There is variability in the degree of denatonium-induced bacterial killing between patients. In CRS with nasal polyps (CRSwNP), patients with increased bacterial killing after stimulation with low levels of denatonium undergo significantly more functional endoscopic sinus surgeries (FESSs) (p = 0.037) and have worse 6-month post-FESS 22-item Sino-Nasal Outcome Test (SNOT-22) scores (p = 0.012). Conclusion Bacterial killing after stimulation with low levels of denatonium correlates with number of prior FESS and postoperative SNOT-22 scores in CRSwNP. Some symptoms of CRS in patients with hyperresponsiveness to low levels of denatonium may be due to increased airway immune activity or inherent disease severity.

Background: Denatonium benzoate (DB) has been shown to influence ongoing ingestive behavior and gut peptide secretion. Objective: We studied how the intragastric administration of DB affects interdigestive motility, motilin and ghrelin plasma concentrations, hunger and satiety ratings, and food intake in healthy volunteers. Design: Lingual bitter taste sensitivity was tested with the use of 6 concentrations of DB in 65 subjects. A placebo or 1 μmol DB/kg was given intragastrically to assess its effect on fasting gastrointestinal motility and hunger ratings, motilin and ghrelin plasma concentrations, satiety, and caloric intake. Results: Women (n = 39) were more sensitive toward a lingual bitter stimulus (P = 0.005) than men (n = 26). In women (n = 10), intragastric DB switched the origin of phase III contractions from the stomach to the duodenum (P = 0.001) and decreased hunger ratings (P = 0.04). These effects were not observed in men (n = 10). In women (n = 12), motilin (P = 0.04) plasma concentrations decreased after intragastric DB administration, whereas total and octanoylated ghrelin were not affected. The intragastric administration of DB decreased hunger (P = 0.008) and increased satiety ratings (P = 0.01) after a meal (500 kcal) in 13 women without affecting gastric emptying in 6 women. Caloric intake tended to decrease after DB administration compared with the placebo (mean ± SEM: 720 ± 58 compared with 796 ± 45 kcal; P = 0.08) in 20 women. Conclusions: Intragastric DB administration decreases both antral motility and hunger ratings during the fasting state, possibly because of a decrease in motilin release. Moreover, DB decreases hunger and increases satiety ratings after a meal and shows potential for decreasing caloric intake