Role of the bitter taste receptor T2R38 in upper respiratory infection and chronic rhinosinusitis

Purpose of reviewTaste receptor family 2 (T2R) bitter taste receptors were originally identified and named on the basis of their role in type 2 taste cells of the tongue, in which they serve to detect the presence of potentially harmful ingested chemicals. In 2009, researchers demonstrated that airway epithelial cells also express T2R receptors, but their role in airway physiology and human disease has only recently begun to be identified. Recent findingsRecent research has demonstrated that at least one airway T2R receptor, taste receptor family 2 isoform 38 protein (T2R38) is activated by secreted bacterial products. Activation of T2R38 in sinonasal epithelial cells stimulates nitric oxide production, increasing ciliary beating and directly killing bacteria. Clinical studies have also found correlations of TAS2R38 genotype with susceptibility to gram-negative upper respiratory infection and established T2R38 as an independent risk factor for chronic rhinosinusitis requiring sinus surgery. SummaryThese recent studies identify a role for T2R38 in sinonasal innate immunity and chronic rhinosinusitis. Clinical implications include the potential development of T2R38-directed topical therapies, as well as using taste testing and/or genotyping to predict susceptibility to infection. Further studies are needed to more clearly determine how TAS2R38 genotype affects patient outcomes in chronic rhinosinusitis and other upper airway diseases.

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title: The Missing Sense Modality: The Immune System
abstract: The five senses were handed down by Aristotle. I argue that it has only taken two millennia to recognize that the immune system has been the hidden sensory modality. The immune system completes the range of operation allowing detection of meaningful entities at all distances, from very near to very far. It also withstands the often implicit criteria for being a sense modality. Finally, cross-modal interactions between the immune system and vision and other sense modalities should be possible, opening up new research directions.

title: Oral and extra-oral taste perception.
abstract: Of the five basic taste qualities, the molecular mechanisms underlying sweet, bitter, and umami (savory) taste perception have been extensively elucidated, including the taste receptors and downstream signal transduction molecules. Recent studies have revealed that these taste-related molecules play important roles not only in the oral cavity but also in a variety of tissues including the respiratory tract, stomach, intestines, pancreas, liver, kidney, testes, and brain. This review covers the current knowledge regarding the physiological roles of taste-related molecules in the oral and extra-oral tissues.

title: The bad taste of medicines: overview of basic research on bitter taste.
abstract: Many active pharmaceutical ingredients taste bitter and thus are aversive to children as well as many adults. Encapsulation of the medicine in pill or tablet form, an effective method for adults to avoid the unpleasant taste, is problematic for children. Many children cannot or will not swallow solid dose forms.



This review highlights basic principles of gustatory function, with a special focus on the science of bitter taste, derived from studies of animal models and human psychophysics. We focus on the set of genes that encode the proteins that function as bitter receptors as well as the cascade of events that leads to multidimensional aspects of taste function, highlighting the role that animal models played in these discoveries. We also summarize psychophysical approaches to studying bitter taste in adult and pediatric populations, highlighting evidence of the similarities and differences in bitter taste perception and acceptance between adults and children and drawing on useful strategies from animal models.



Medicine often tastes bitter, and because children are more bitter-sensitive than are adults, this creates problems with compliance. Bitter arises from stimulating receptors in taste receptor cells, with signals processed in the taste bud and relayed to the brain. However, there are many gaps in our understanding of how best to measure bitterness and how to ameliorate it, including whether it is more efficiently addressed at the level of receptor and sensory signaling, at the level of central processing, or by masking techniques. All methods of measuring responsiveness to bitter ligands-in animal models through human psychophysics or with "electronic tongues"-have limitations.



Better-tasting medications may enhance pediatric adherence to drug therapy. Sugars, acids, salt, and other substances reduce perceived bitterness of several pharmaceuticals, and although pleasant flavorings may help children consume some medicines, they often are not effective in suppressing bitter tastes. Further development of psychophysical tools for children will help us better understand their sensory worlds. Multiple testing strategies will help us refine methods to assess acceptance and compliance by various pediatric populations. Research involving animal models, in which the gustatory system can be more invasively manipulated, can elucidate mechanisms, ultimately providing potential targets. These approaches, combined with new technologies and guided by findings from clinical studies, will potentially lead to effective ways to enhance drug acceptance and compliance in pediatric populations.

title: Solitary chemoreceptor cells in the nasal cavity serve as sentinels of respiration
abstract: Inhalation of irritating substances leads to activation of the trigeminal nerve, triggering protective reflexes that include apnea or sneezing. Receptors for trigeminal irritants are generally assumed to be located exclusively on free nerve endings within the nasal epithelium, requiring that trigeminal irritants diffuse through the junctional barrier at the epithelial surface to activate receptors. We find, in both rats and mice, an extensive population of chemosensory cells that reach the surface of the nasal epithelium and form synaptic contacts with trigeminal afferent nerve fibers. These chemosensory cells express T2R “bitter-taste” receptors and α-gustducin, a G protein involved in chemosensory transduction. Functional studies indicate that bitter substances applied to the nasal epithelium activate the trigeminal nerve and evoke changes in respiratory rate. By extending to the surface of the nasal epithelium, these chemosensory cells serve to expand the repertoire of compounds that can activate trigeminal protective reflexes. The trigeminal chemoreceptor cells are likely to be remnants of the phylogenetically ancient population of solitary chemoreceptor cells found in the epithelium of all anamniote aquatic vertebrates.

title: Nasal solitary chemoreceptor cell responses to bitter and trigeminal stimulants in vitro.
abstract: Nasal trigeminal chemosensitivity in mice and rats is mediated in part by epithelial solitary chemoreceptor (chemosensory) cells (SCCs), but the exact role of these cells in chemoreception is unclear. Histological evidence suggests that SCCs express elements of the bitter taste transduction pathway including T2R (bitter taste) receptors, the G protein alpha-gustducin, PLCbeta2, and TRPM5, leading to speculation that SCCs are the receptor cells that mediate trigeminal nerve responses to bitter taste receptor ligands. To test this hypothesis, we used calcium imaging to determine whether SCCs respond to classic bitter-tasting or trigeminal stimulants. SCCs from the anterior nasal cavity were isolated from transgenic mice in which green fluorescent protein (GFP) expression was driven by either TRPM5 or gustducin. Isolated cells were exposed to a variety of test stimuli to determine which substances caused an increase in intracellular Ca2+ ([Ca2+]i). GFP-positive cells respond with increased [Ca2+]i to the bitter receptor ligand denatonium and this response is blocked by the PLC inhibitor U73122. In addition, GFP+ cells respond to the neuromodulators adenosine 5'-triphosphate and acetylcholine but only very rarely to other bitter-tasting or trigeminal stimuli. Our results demonstrate that TRPM5- and gustducin-expressing nasal SCCs respond to the T2R agonist denatonium via a PLC-coupled transduction cascade typical of T2Rs in the taste system.

title: Cholinergic neurotransmission links solitary chemosensory cells to nasal inflammation
abstract: Millions of people worldwide suffer from chronic nasal inflammation involving obstructed airflow and nasal discharge. Although nasal inflammation is often considered to be a reaction to allergens, approximately one-quarter of all cases are nonallergic rhinitis. The causes of this disease are unknown, but symptoms may be triggered or exacerbated by a variety of inhaled irritants or even seemingly innocuous odors. We report here that specialized chemosensory cells of the nasal epithelium of mice detect potential irritants and transmit this information to pain-sensing nerve terminals, which then release bioactive peptides to trigger an inflammatory response—all without the necessity for activity of the adaptive immune system. This previously unidentified pathway may offer therapeutic targets for intervention in nonallergic rhinitis. Solitary chemosensory cells (SCCs) of the nasal cavity are specialized epithelial chemosensors that respond to irritants through the canonical taste transduction cascade involving Gα-gustducin and transient receptor potential melastatin 5. When stimulated, SCCs trigger peptidergic nociceptive (or pain) nerve fibers, causing an alteration of the respiratory rate indicative of trigeminal activation. Direct chemical excitation of trigeminal pain fibers by capsaicin evokes neurogenic inflammation in the surrounding epithelium. In the current study, we test whether activation of nasal SCCs can trigger similar local inflammatory responses, specifically mast cell degranulation and plasma leakage. The prototypical bitter compound, denatonium, a well-established activator of SCCs, caused significant inflammatory responses in WT mice but not mice with a genetic deletion of elements of the canonical taste transduction cascade, showing that activation of taste signaling components is sufficient to trigger local inflammation. Chemical ablation of peptidergic trigeminal fibers prevented the SCC-induced nasal inflammation, indicating that SCCs evoke inflammation only by neural activity and not by release of local inflammatory mediators. Additionally, blocking nicotinic, but not muscarinic, acetylcholine receptors prevents SCC-mediated neurogenic inflammation for both denatonium and the bacterial signaling molecule 3-oxo-C12-homoserine lactone, showing the necessity for cholinergic transmission. Finally, we show that the neurokinin 1 receptor for substance P is required for SCC-mediated inflammation, suggesting that release of substance P from nerve fibers triggers the inflammatory events. Taken together, these results show that SCCs use cholinergic neurotransmission to trigger peptidergic trigeminal nociceptors, which link SCCs to the neurogenic inflammatory pathway.

title: Host-microbial interactions in patients with chronic rhinosinusitis.
abstract: Credit can now be obtained, free for a limited time, by reading the review articles in this issue. Please note the instructions listed below: 1. Review the target audience, learning objectives and author disclosures. 2. Complete the pre-test online at www.jacionline.org (click on the Online CME heading). 3. Follow the online instructions to read the full version of the article, including the clinical vignette and review components. 4. Complete the post-test. At this time, you will have earned 1.00 AMA PRA Category 1 CME Credit. 5. Approximately 4 weeks later you will receive an online assessment regarding your application of this article to your practice. Once you have completed this assessment, you will be eligible to receive 2 MOC Part II Self-Assessment credits from the American Board of Allergy and Immunology.

title: Innate immunity in the respiratory epithelium.
abstract: The airway epithelium represents the first point of contact for inhaled foreign organisms. The protective arsenal of the airway epithelium is provided in the form of physical barriers and a vast array of receptors and antimicrobial compounds that constitute the innate immune system. Many of the known innate immune receptors, including the Toll-like receptors and nucleotide oligomerization domain-like receptors, are expressed by the airway epithelium, which leads to the production of proinflammatory cytokines and chemokines that affect microorganisms directly and recruit immune cells, such as neutrophils and T cells, to the site of infection. The airway epithelium also produces a number of resident antimicrobial proteins, such as lysozyme, lactoferrin, and mucins, as well as a swathe of cationic proteins. Dysregulation of the airway epithelial innate immune system is associated with a number of medical conditions that can result in compromised immunity and chronic inflammation of the lung. This review focuses on the innate immune capabilities of the airway epithelium and its role in protecting the lung from infection as well as the outcomes when its function is compromised.

title: Sinonasal Mucociliary Clearance in Health and Disease
abstract: Although much has been elucidated in the past 170 years concerning the precise mechanism of ciliary function in the healthy or diseased human respiratory system, significant questions remain. The first description of ciliary action is credited to Sharpey in 1835. However, the importance of mucosal function was not apparent until Hilding's investigations of the postsurgical canine sinus demonstrated scar formation and disruption of mucociliary clearance. Subsequently, several techniques for mucosal coverage of exposed bone, most notably by Sewall and Boyden, were reported. The underlying physiology explaining the importance of the mucosa and the concept of mucosal preservation became apparent with the description of the sinonasal mucociliary flow patterns by Messerklinger; and thus the restoration of natural sinus physiology, ie, mucociliary clearance, became the goal of both medical and surgical treatment of sinonasal inflammatory disease. Clearance of benign and pathological substances in the mucus is governed by the propulsive force of the beating cilia and the physical characteristics of the overlying mucus. The respiratory cilia continually beat in a coordinated fashion, and in times of stress (eg, exercise, infection, or fever) ciliary beat frequency increases to accelerate mucus clearance. Thus, upper airway ciliary motility is under dynamic modulation. Multiple investigations incontrovertibly demonstrate a marked decrease in sinonasal mucociliary clearance in patients with chronic rhinosinusitis. Possible explanations for this finding are 1) a reduced basal ciliary beat frequency, 2) an alteration of the viscoelastic properties of airway secretions, and/or 3) a blunted dynamic response of sinonasal cilia to environmental stimuli. Studies of the first two explanations yield conflicting results, and to date, the third possibility remains uninvestigated. A review of the current understanding of the cellular regulation of respiratory ciliary activity and its contribution to chronic rhinosinusitis is presented.

title: Airway defense mechanisms.
abstract: A multitude of overlapping defenses has evolved to help combat the inventiveness of pathogens seeking to invade us, and because the lung is the most common primary route of infection, it is in the lung that the most varied responses are seen. This article focuses on recent research, particularly in innate immunity, and gives a general overview of the defense systems so far identified. Of particular interest is the markedly increased understanding of the role of small molecules such as defensins, cathelicidins, and collectins. Areas in which abnormalities may be relevant to patients with bronchiectasis are highlighted.

title: Epithelium, cilia, and mucus: their importance in chronic rhinosinusitis.
abstract: Chronic rhinosinusitis is a common disease resulting from inflammation of the sinonasal mucosa. It has long been recognized that patients with chronic rhinosinusitis have impaired capacity to clear sinonasal secretions. However, the cause of this pathologic process is not well understood. In this article the components of mucociliary clearance, including cilia, mucus production, and cilia beat frequency, are reviewed and alterations of the system discussed regarding contribution to the disease process.

title: Acquired cilia dysfunction in chronic rhinosinusitis
abstract: Cilia are complex and powerful cellular structures of the respiratory mucosa that play a critical role in airway defense. Respiratory epithelium is lined with cilia that perform an integrated and coordinated mechanism called mucociliary clearance. Mucociliary clearance is the process by which cilia transport the mucus blanket overlying respiratory mucosa to the gastrointestinal tract for ingestion. It is the primary means by which the airway clears pathogens, allergens, debris, and toxins. The complex structure and regulatory mechanisms that dictate the form and function of normal cilia are not entirely understood, but it is clear that ciliary dysfunction results in impaired respiratory defense.  A literature review of the current knowledge of cilia dysfunction in chronic rhinosinsusitis was conducted.  Ciliary dysfunction may be primary, the result of genetic mutations resulting in abnormal cilia structure, or, more commonly, secondary, the result of environmental, infectious, or inflammatory stimuli that disrupt normal motility or coordination. Patients with chronic rhinosinusitis (CRS) have been found to have impaired mucociliary clearance. Many biochemical, environmental, and mechanical stimuli have been shown to influence ciliary beat frequency, and common microbial pathogens of respiratory mucosa such as Pseudomonas aeruginosa and Haemophilus influenzae have developed toxins that appear to interrupt normal mucociliary function. Furthermore, inflammatory mediators known to be present in patients with CRS appear to impair secondarily mucociliary clearance.  The goal of this article is to summarize the recent developments in the understanding of cilia dysfunction and mucociliary clearance in CRS.

title: Chapter 4: Chronic rhinosinusitis.
abstract: Chronic rhinosinusitis (CRS) is the second most common chronic medical condition in the United States. It represents a group of disorders characterized by inflammation of the nasal mucosa and paranasal sinuses of at least 12 weeks duration. CRS with or without nasal polyps is defined as inflammation of the nose characterized by two or more symptoms, one of which should be either nasal blockage, obstruction, congestion, or nasal discharge (anterior/posterior nasal drip); with or without facial pain/pressure; and/or with or without reduction or loss of smell. Symptomatology should be supported by obvious disease evident in either nasal endoscopy or computed tomography imaging. Although CRS is not likely to be cured by either medical or surgical therapy, it can generally be controlled. Best medical evidence supports maintenance therapy with intranasal corticosteroids and saline irrigation. For exacerbations, short to intermediate courses of antibiotics (up to 4-weeks) with or without oral corticosteroids are recommended. For patients with difficult-to-treat CRS, functional endoscopic sinus surgery provides an adjunctive therapeutic option.

title: Chapter 13: Olfactory disorders.
abstract: Decreased sense of smell can lead to significant impairment of quality of life, including taste disturbance and loss of pleasure from eating with resulting changes in weight, and difficulty in avoiding health risks such as spoiled food or leaking natural gas. Recent epidemiological reports have shown that despite fairly low self-reported prevalence of these disorders in large population studies, when validated smell identification or threshold tests are used they reveal quite a high prevalence of hyposmia and anosmia in certain groups, especially the elderly. Several different pathophysiological processes, such as head trauma, aging, autoimmunity, and toxic exposures, can contribute to smell impairment, with distinct implications concerning prognosis and possible treatment. Otolaryngologists are most likely to see this symptom in patients with chronic rhinosinusitis, and this now appears to be caused more by the mucosal inflammation than by physical airway obstruction.

title: Bacteriology and antibiotic resistance in chronic rhinosinusitis.
abstract: Chronic rhinosinusitis (CRS) is a prevalent health care problem that may be commonly encountered in patients desiring aesthetic or reconstructive rhinoplasty. The purpose of this article is to review the common bacterial pathogens associated with CRS, as well as patterns of bacterial resistance in this patient subset. Close understanding of microbial pathogens involved in CRS and their associated resistance patterns will guide facial plastic surgeons in optimally managing this important potential comorbidity, and in turn positively influence the outcome of rhinoplasty.

title: Anatomy and physiology of the upper airway.
abstract: The nose is the major portal of air exchange between the internal and external environment. The nose participates in the vital functions of conditioning inspired air toward a temperature of 37°C and 100% relative humidity, providing local defense and filtering inhaled particulate matter and gases. It also functions in olfaction, which provides both a defense and pleasure for the individual. Understanding normal physiology provides the basis for recognizing abnormalities.

title: Chapter 1: Sinonasal anatomy and function.
abstract: An understanding of paranasal sinus anatomy based on important fixed landmarks rather than variable anatomy is critical to ensure safe and complete surgery. The concept of the paranasal surgical box defines the anatomic limits of dissection. The boundaries of the surgical box include the middle turbinate medially, orbital wall laterally, and skull base superiorly. The "vertical component" of the surgical box defines the boundaries of the frontal recess and includes the middle turbinate and intersinus septum medially, medial orbital wall and orbital roof laterally, nasofrontal beak anteriorly, and skull base and posterior table of frontal sinus posteriorly. The paranasal sinuses are divided into anterior, posterior, and sphenoidal functional cavities based on their distinct drainage pathways into the nose. The ultimate goal of surgery is to create a functional sinus cavity. Application of the paranasal surgical box and its vertical component enables the surgeon to view the limits of dissection with a single position of the endoscope. This will ensure complete dissection of the functional sinonasal compartments and effectively avoid leaving behind disconnected cells from the surgical cavity, mucocele formation, mucous recirculation, overcome obstructive phenomenon and enable maximal delivery of topical therapy in the post-operative setting. This article reviews the structure and function of the nasal cartilages and turbinates. It also describes the concept of the paranasal surgical box, key anatomical landmarks and limits of dissection. Normal anatomy and common variants of normal anatomy are discussed.

title: Nitric oxide and antimicrobial activity of reactive oxygen intermediates.
abstract: It is well documented that nitric oxide contributes to the bactericidal activities of phagocytes. Murine activated neutrophils and macrophages produce both reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). However, only neutrophils in the presence of myeloperoxidase, produce an antimicrobial agent, hypochlorous acid (HOCl). Complex interactions of RNI (nitric oxide) with other antimicrobial agents of phagocytes are likely to exist, but these have not been clearly demonstrated. In this study, we treated bacteria (Escherichia coli) with the NO donor, S-nitrosoglutathione (GSNO) and hydrogen peroxide (H2O2) or HOCl. We found that exposure to H2O2 of the bacteria tested resulted in minimal toxicity. However the killing activities of H2O2 were potentiated by GSNO. On the contrary, the NO-donor completely abolished the bactericidal activity of HOCl. Our results indicate that NO-donating drugs in non toxic concentrations used for experimental purposes may strongly affect the cytotoxic activity of neutrophils and macrophages. We suggest that the similar interactions may exist at sites of inflammation.

title: Bacterial Pattern in Chronic Sinusitis and Cystic Fibrosis
abstract: To assess the association between pathogenic bacteria found in bronchoalveolar lavages and paranasal cavity cultures in patients with cystic fibrosis (CF) who underwent endonasal endoscopic sinus surgery. The authors hypothesized that the pathogenic bacterial pattern of the upper airway would be associated with that of the lower airway.  Prospective case series with planned data collection.  Tertiary referral center.  A group of 16 patients with diagnosis of CF who underwent endonasal endoscopic sinus surgery from July 2001 to August 2009 participated in the study. Culture samples were obtained from bronchoalveolar lavages and paranasal cavities. A Fisher exact test was performed to examine the significance of the association between upper airway and lower airway cultures.  The most frequent microorganisms in cultures of para-nasal cavities and bronchoalveolar lavages were Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus viridans. Statistical analysis revealed a significant association between upper airway cultures (paranasal cavities) and lower airway cavities (bronchoalveolar lavages) for the 2 most frequent microorganisms, P aeruginosa and S aureus (P = .007 and P = .030, respectively).  The authors confirmed their initial hypothesis that a significant association between bronchoalveolar lavages and sinus cultures was established, once more confirming the idea of a unified airway. They found chronic rhinosinusitis with polyps to be a common clinical presentation in patients with CF. Further studies are required to indicate the role of antibiotics and the pathogenesis of the microorganisms as a manifestation of clinical severity.

title: Retrospective analysis of chronic rhinosinusitis in patients with cystic fibrosis.
abstract: Chronic rhinosinusitis is very common in patients with cystic fibrosis. This can be explained by the unified airway concept, where the same pathophysiological phenomenon that affects the lungs, affects the paranasal sinuses. The management of these cases is difficult.



To describe the teamwork of otolaryngologists and bronchopulmonary specialists in patients with cystic fibrosis.



We performed a descriptive, retrospective study over the last 17 years, which included 14 patients with chronic rhinosinusitis and cystic fibrosis attended at a private hospital.



Of the patients, 64% were male and the median age was 23 years. The most frequent mutations found were ΔF508, M470 and R553. All of the patients with ΔF508 mutation had nasal polyps. 100% of the patients had clinical findings of chronic rhinosinusitis. All the patients had had endoscopic nasal surgery. The median number of endoscopic surgeries was 2.



Given the high prevalence of chronic rhinosinusitis in patients with cystic fibrosis, everyone should have a computed tomography scan of the paranasal sinuses during the initial assessment, considering that sinus germs are the ones that colonise the lower airway. The otolaryngologist should be part of the cystic fibrosis team. Before receiving a lung transplant or in cases of chronic headache, endoscopic surgery should be performed in patients in whom medical treatment fails to clear the sinuses because this infection is the one that colonises the lower airway.

title: Extensive surgical and comprehensive postoperative medical management for cystic fibrosis chronic rhinosinusitis
abstract: Chronic rhinosinusitis has a major impact on the quality of life of patients with cystic fibrosis (CF) and may contribute to progression of chronic lung disease. Despite multiple sinus surgeries, maxillary sinus involvement is a recurrent problem. The modified endoscopic medial maxillectomy (MEMM) permits debridement in the clinic, improves mucus clearance with nasal irrigations, and increases access for topical delivery of therapeutics. However, clinical outcomes of aggressive sinus surgery with regimented postoperative medical treatment have not been systematically evaluated.  CF patients completed the 22-Item Sinonasal Outcome Test questionnaires before sinus surgery (and bilateral MEMM) and at sequential postoperative visits.  Lund-Kennedy endoscopic score and pulmonary function tests (forced expiratory volume at 1 second percent [FEV1%] predicted). Culture-directed antibiotic therapy, prednisone, and topical irrigations were initiated postoperatively.  Twenty-two patients (mean age, 26.5 years; 4.9 prior sinus operations) underwent MEMM and sinus surgery. Symptom scores were significantly reduced at 60 days (primary outcome, 64.7 ± 18.4 presurgery versus 27.5 ± 15.3 postsurgery; p < 0.0001) and up to a year postoperatively (27.6 ± 12.6; p < 0.0001). Endoscopic scores were also reduced after surgery (10.4 ± 1.1 presurgery versus 5.7 ± 2.4 [30 days], 5.7 ± 1.4 [60 days], 5.8 ± 1.3 [120 days], and 6.0 ± 1.1 [1 year]; p < 0.0001)]. There were no differences in FEV1% predicted up to 1 year postoperatively, but hospital admissions secondary to pulmonary exacerbations significantly decreased (2.0 ± 1.4 versus 3.2 ± 2.4, respectively; p < 0.05).  Prospective evaluation indicates sinus surgery with MEMM is associated with marked improvement in sinus disease outcomes. Additional studies are necessary to confirm whether this treatment paradigm is associated with improved CF pulmonary disease.

title: Taste receptor signalling – from tongues to lungs
abstract: Taste buds are the transducing endorgans of gustation. Each taste bud comprises 50–100 elongated cells, which extend from the basal lamina to the surface of the tongue, where their apical microvilli encounter taste stimuli in the oral cavity. Salts and acids utilize apically located ion channels for transduction, while bitter, sweet and umami (glutamate) stimuli utilize G‐protein‐coupled receptors (GPCRs) and second‐messenger signalling mechanisms. This review will focus on GPCR signalling mechanisms. Two classes of taste GPCRs have been identified, the T1Rs for sweet and umami (glutamate) stimuli and the T2Rs for bitter stimuli. These low affinity GPCRs all couple to the same downstream signalling effectors that include Gβγ activation of phospholipase Cβ2, 1,4,5‐inositol trisphosphate mediated release of Ca2+ from intracellular stores and Ca2+‐dependent activation of the monovalent selective cation channel, TrpM5. These events lead to membrane depolarization, action potentials and release of ATP as a transmitter to activate gustatory afferents. The Gα subunit, α‐gustducin, activates a phosphodiesterase to decrease intracellular cAMP levels, although the precise targets of cAMP have not been identified. With the molecular identification of the taste GPCRs, it has become clear that taste signalling is not limited to taste buds, but occurs in many cell types of the airways. These include solitary chemosensory cells, ciliated epithelial cells and smooth muscle cells. Bitter receptors are most abundantly expressed in the airways, where they respond to irritating chemicals and promote protective airway reflexes, utilizing the same downstream signalling effectors as taste cells.

title: Taste perception: from the tongue to the testis.
abstract: In mammals, the sense of taste helps in the evaluation and consumption of nutrients, and in avoiding toxic substances and indigestible materials. Distinct cell types expressing unique receptors detect each of the five basic tastes: salty, sour, bitter, sweet and umami. The latter three tastes are detected by two distinct families of G protein-coupled receptors: T2Rs and T1Rs. Interestingly, these taste receptors have been found in tissues other than the tongue, such as the digestive system, respiratory system, brain, testis and spermatozoa. The functional implications of taste receptors distributed throughout the body are unknown. We therefore reviewed the remarkable advances in our understanding of the molecular basis of taste perception in 'taste' and 'non-taste' tissues. We also present our speculations on the direction of further research in the field of male reproduction.

title: Chemosensors in the nose: guardians of the airways.
abstract: The G-protein-coupled receptor molecules and downstream effectors that are used by taste buds to detect sweet, bitter, and savory tastes are also utilized by chemoresponsive cells of the airways to detect irritants. Here, we describe the different cell types in the airways that utilize taste-receptor signaling to trigger protective epithelial and neural responses to potentially dangerous toxins and bacterial infection.

title: Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals
abstract: The upper respiratory tract is continually assaulted with harmful dusts and xenobiotics carried on the incoming airstream. Detection of such irritants by the trigeminal nerve evokes protective reflexes, including sneezing, apnea, and local neurogenic inflammation of the mucosa. Although free intra-epithelial nerve endings can detect certain lipophilic irritants (e.g., mints, ammonia), the epithelium also houses a population of trigeminally innervated solitary chemosensory cells (SCCs) that express T2R bitter taste receptors along with their downstream signaling components. These SCCs have been postulated to enhance the chemoresponsive capabilities of the trigeminal irritant-detection system. Here we show that transduction by the intranasal solitary chemosensory cells is necessary to evoke trigeminally mediated reflex reactions to some irritants including acyl–homoserine lactone bacterial quorum-sensing molecules, which activate the downstream signaling effectors associated with bitter taste transduction. Isolated nasal chemosensory cells respond to the classic bitter ligand denatonium as well as to the bacterial signals by increasing intracellular Ca2+. Furthermore, these same substances evoke changes in respiration indicative of trigeminal activation. Genetic ablation of either Gα-gustducin or TrpM5, essential elements of the T2R transduction cascade, eliminates the trigeminal response. Because acyl–homoserine lactones serve as quorum-sensing molecules for Gram-negative pathogenic bacteria, detection of these substances by airway chemoreceptors offers a means by which the airway epithelium may trigger an epithelial inflammatory response before the bacteria reach population densities capable of forming destructive biofilms.

title: A second N-acylhomoserine lactone signal produced by Pseudomonas aeruginosa.
abstract: Quorum sensing systems are used by a number of Gram-negative bacterial species to regulate specific sets of genes in a cell density-dependent manner. Quorum sensing involves synthesis and detection of extracellular signals termed autoinducers. As shown in recombinant Escherichia coli, the Pseudomonas aeruginosa autoinducer (PAI) N-(3-oxododecanoyl)homoserine lactone, together with the lasR gene product, activate the P. aeruginosa lasB gene. In this study, PAI was shown to activate lasB-lacZ expression in a P. aeruginosa lasR mutant containing a plasmid with lasR under the control of the lac promoter. The concentration of PAI necessary for half-maximal activation of the lasB-lacZ fusion was approximately 1 microM, which is within the range of PAI levels found in P. aeruginosa culture fluids. The effect of PAI on a P. aeruginosa lasR mutant containing a plasmid with lasR under the control of its own promoter and containing the lasB-lacZ fusion was also tested. Although extracts of culture fluid activated the lasB promoter in this construct, concentrations of PAI as high as 10 microM did not. This indicates the presence of a second extracellular factor (factor 2) that is required for lasB activation in P. aeruginosa when lasR is controlled by its own promoter but not when lasR is controlled by a strong foreign promoter. Factor 2 was shown to be N-butyrylhomoserine lactone. Although recombinant E. coli cells containing the PAI synthase gene, lasI, produce PAI, these cells do not produce factor 2. Furthermore, a P. aeruginosa mutant that produced about 0.1% of the wild-type level of PAI made about 5% of the wild-type level of factor 2. This indicates that factor 2 synthesis results from the activity of a gene product other than PAI synthase. The role of factor 2 in virulence gene regulation remains to be determined, but this compound may affect the expression of lasR, which in turn activates transcription of numerous virulence genes in the presence of sufficient PAI. Apparently, multiple quorum sensing systems can occur and interact with each other in a single bacterial species.

title: The Multiple Signaling Systems Regulating Virulence in Pseudomonas aeruginosa
abstract: Cell-to-cell communication is a major process that allows bacteria to sense and coordinately react to the fluctuating conditions of the surrounding environment. In several pathogens, this process triggers the production of virulence factors and/or a switch in bacterial lifestyle that is a major determining factor in the outcome and severity of the infection. Understanding how bacteria control these signaling systems is crucial to the development of novel antimicrobial agents capable of reducing virulence while allowing the immune system of the host to clear bacterial infection, an approach likely to reduce the selective pressures for development of resistance. We provide here an up-to-date overview of the molecular basis and physiological implications of cell-to-cell signaling systems in Gram-negative bacteria, focusing on the well-studied bacterium Pseudomonas aeruginosa. All of the known cell-to-cell signaling systems in this bacterium are described, from the most-studied systems, i.e., N-acyl homoserine lactones (AHLs), the 4-quinolones, the global activator of antibiotic and cyanide synthesis (GAC), the cyclic di-GMP (c-di-GMP) and cyclic AMP (cAMP) systems, and the alarmones guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), to less-well-studied signaling molecules, including diketopiperazines, fatty acids (diffusible signal factor [DSF]-like factors), pyoverdine, and pyocyanin. This overview clearly illustrates that bacterial communication is far more complex than initially thought and delivers a clear distinction between signals that are quorum sensing dependent and those relying on alternative factors for their production.

title: Sesquiterpenoids Lactones: Benefits to Plants and People
abstract: Sesquiterpenoids, and specifically sesquiterpene lactones from Asteraceae, may play a highly significant role in human health, both as part of a balanced diet and as pharmaceutical agents, due to their potential for the treatment of cardiovascular disease and cancer. This review highlights the role of sesquiterpene lactones endogenously in the plants that produce them, and explores mechanisms by which they interact in animal and human consumers of these plants. Several mechanisms are proposed for the reduction of inflammation and tumorigenesis at potentially achievable levels in humans. Plants can be classified by their specific array of produced sesquiterpene lactones, showing high levels of translational control. Studies of folk medicines implicate sesquiterpene lactones as the active ingredient in many treatments for other ailments such as diarrhea, burns, influenza, and neurodegradation. In addition to the anti-inflammatory response, sesquiterpene lactones have been found to sensitize tumor cells to conventional drug treatments. This review explores the varied ecological roles of sesquiterpenes in the plant producer, depending upon the plant and the compound. These include allelopathy with other plants, insects, and microbes, thereby causing behavioural or developmental modification to these secondary organisms to the benefit of the sesquiterpenoid producer. Some sesquiterpenoid lactones are antimicrobial, disrupting the cell wall of fungi and invasive bacteria, whereas others protect the plant from environmental stresses that would otherwise cause oxidative damage. Many of the compounds are effective due to their bitter flavor, which has obvious implications for human consumers. The implications of sesquiterpenoid lactone qualities for future crop production are discussed.

title: Worldwide haplotype diversity and coding sequence variation at human bitter taste receptor loci
abstract: Bitter taste perception in humans is mediated by receptors encoded by 25 genes that together comprise the TAS2R (or T2R) gene family. The ability to identify the ligand(s) for each of these receptors is dependent on understanding allelic variation in TAS2R genes, which may have a significant effect on ligand recognition. To investigate the extent of coding variation among TAS2R alleles, we performed a comprehensive evaluation of sequence and haplotype variation in the human bitter taste receptor gene repertoire. We found that these genes exhibit substantial coding sequence diversity. In a worldwide population sample of 55 individuals, we found an average of 4.2 variant amino acid positions per gene. In aggregate, the 24 genes analyzed here, along with the phenylthiocarbamide (PTC) receptor gene analyzed previously, specify 151 different protein coding haplotypes. Analyses of the ratio of synonymous and nonsynonymous nucleotide substitutions using the Ka/Ks ratio revealed an excess of amino acid substitutions relative to most other genes examined to date (Ka/Ks=0.94). In addition, comparisons with more than 1,500 other genes revealed that levels of diversity in the TAS2R genes were significantly greater than expected (π=0.11%; p<0.01), as were levels of differentiation among continental populations (FST=0.22; p<0.05). These diversity patterns indicate that unusually high levels of allelic variation are found within TAS2R loci and that human populations differ appreciably with respect to TAS2R allele frequencies. Diversity in the TAS2R genes may be accounted for by natural selection, which may have favored alleles responsive to toxic, bitter compounds found in plants. These findings are consistent with the view that different alleles of the TAS2R genes encode receptors that recognize different ligands, and suggest that the haplotypes we have identified will be important in studies of receptor‐ligand recognition.  Mutat 26(3), 199–204, 2005.

title: Diet shapes the evolution of the vertebrate bitter taste receptor gene repertoire.
abstract: Vertebrate Tas2r taste receptors bind to bitter compounds, which are typically poisonous, to elicit bitter sensation to prevent the ingestion of toxins. Previous studies noted a marked variation in the number of Tas2r genes among species, but the underlying cause is unclear. To address this question, we compile the Tas2r gene repertoires from 41 mammals, 4 birds, 2 reptiles, 1 amphibian, and 6 fishes. The number of intact Tas2r genes varies from 0 in the bottlenose dolphin to 51 in the Western clawed frog, with numerous expansions and contractions of the gene family throughout vertebrates, especially among tetrapods. The Tas2r gene number in a species correlates with the fraction of plants in its diet. Because plant tissues contain more toxic compounds than animal tissues do, our observation supports the hypothesis that dietary toxins are a major selective force shaping the diversity of the Tas2r repertoire.

title: Allelic variation in TAS2R bitter receptor genes associates with variation in sensations from and ingestive behaviors toward common bitter beverages in adults.
abstract: The 25 human bitter receptors and their respective genes (TAS2Rs) contain unusually high levels of allelic variation, which may influence response to bitter compounds in the food supply. Phenotypes based on the perceived bitterness of single bitter compounds were first linked to food preference over 50 years ago. The most studied phenotype is propylthiouracil bitterness, which is mediated primarily by the TAS2R38 gene and possibly others. In a laboratory-based study, we tested for associations between TAS2R variants and sensations, liking, or intake of bitter beverages among healthy adults who were primarily of European ancestry. A haploblock across TAS2R3, TAS2R4, and TAS2R5 explained some variability in the bitterness of espresso coffee. For grapefruit juice, variation at a TAS2R19 single nucleotide polymorphism (SNP) was associated with increased bitterness and decreased liking. An association between a TAS2R16 SNP and alcohol intake was identified, and the putative TAS2R38-alcohol relationship was confirmed, although these polymorphisms did not explain sensory or hedonic responses to sampled scotch whisky. In summary, TAS2R polymorphisms appear to influence the sensations, liking, or intake of common and nutritionally significant beverages. Studying perceptual and behavioral differences in vivo using real foods and beverages may potentially identify polymorphisms related to dietary behavior even in the absence of known ligands.

title: Sweet and bitter tastes of alcoholic beverages mediate alcohol intake in of-age undergraduates
abstract: Alcoholic beverages are complex stimuli, giving rise to sensations that promote or inhibit intake. Previous research has shown associations between 6-n-propylthiouracil (PROP) bitterness, one marker of genetic variation in taste, and alcohol behaviors. We tested the PROP bitterness and alcohol intake relationship as mediated by tastes of sampled alcoholic beverages. Forty-nine undergraduates (mean age=22 years) participated. According to the Alcohol Use Disorders Identification Test (AUDIT), only 3 of 49 subjects reported patterns indicating problematic drinking. Participants used the general Labeled Magnitude Scale to rate PROP bitterness and tastes from and preference for Pilsner beer, blended scotch whiskey, instant espresso and unsweetened grapefruit juice. Alcohol intake was reported over a typical week. Regression analysis tested the hypothesis that PROP bitterness influenced alcohol bitterness and sweetness, which in turn predicted alcohol intake. Those who tasted less PROP bitterness tasted all beverages as less bitter and more preferred. Sweetness of scotch was significantly greater in those who tasted PROP as least bitter. For scotch, greater sweetness and less bitterness from sampled scotch were direct predictors of greater alcohol intake. For beer, preference ratings were better predictors of alcohol intake than the bitter or sweet tastes of the sampled beer. These findings support that PROP bitterness predicts both positive and negative tastes from alcoholic beverages and that those tastes may predict alcohol intake. The college environment may attenuate direct effects of PROP bitterness and intake. Here, PROP bitterness does not predict alcohol intake directly, but acts instead through sweet and bitter tastes of alcoholic beverages.

title: Inflammation-mediated upregulation of centrosomal protein 110, a negative modulator of ciliogenesis, in patients with chronic rhinosinusitis.
abstract: Sinonasal mucosa in patients with chronic rhinosinusitis (CRS) is often devoid of motile cilia. This defect is presumed to result from prolonged inflammation, infection, or both. However, the mechanism underlying this observation is unknown. Recently, centrosomal protein 110 (Cp110) was shown to prevent the terminal step in ciliary maturation (ie, elongation), suggesting that Cp110 might be involved in pathological states in which ciliation is abnormal.



First, we sought to investigate the expression of Cp110 in sinonasal mucosa from patients with CRS and control subjects. Second, we sought to determine the extent that inflammatory cytokines modulate Cp110 expression and ciliary maturation in vitro.



Sinonasal mucosal specimens from patients with and without CRS were analyzed for Cp110 mRNA and protein expression. Furthermore, human and murine nasal respiratory epithelial cultures were used to investigate Cp110 expression under normal growth conditions and in the presence of exogenous proinflammatory cytokines.



Increased Cp110 mRNA and protein expression was found in sinonasal mucosal specimens from patients with CRS compared with that seen in control specimens. During ciliogenesis in vitro, the expression of Cp110 gradually decreased in cultures derived from patients without CRS but remained increased in cultures derived from patients with CRS. Furthermore, cultures grown in the presence of proinflammatory cytokines demonstrated increased levels of Cp110 expression with concomitant inhibition of ciliogenesis.



Increased Cp110 expression in mucosa from patients with CRS might contribute to the poor ciliation observed in patients with CRS. Exogenous cytokine exposure maintains increased levels of Cp110 expression. Regulation of Cp110 expression by inflammation warrants additional investigation because it might offer a novel target in the management of respiratory tract diseases.

title: T2R38 taste receptor polymorphisms underlie susceptibility to upper respiratory infection.
abstract: Innate and adaptive defense mechanisms protect the respiratory system from attack by microbes. Here, we present evidence that the bitter taste receptor T2R38 regulates the mucosal innate defense of the human upper airway. Utilizing immunofluorescent and live cell imaging techniques in polarized primary human sinonasal cells, we demonstrate that T2R38 is expressed in human upper respiratory epithelium and is activated in response to acyl-homoserine lactone quorum-sensing molecules secreted by Pseudomonas aeruginosa and other gram-negative bacteria. Receptor activation regulates calcium-dependent NO production, resulting in stimulation of mucociliary clearance and direct antibacterial effects. Moreover, common polymorphisms of the TAS2R38 gene were linked to significant differences in the ability of upper respiratory cells to clear and kill bacteria. Lastly, TAS2R38 genotype correlated with human sinonasal gram-negative bacterial infection. These data suggest that T2R38 is an upper airway sentinel in innate defense and that genetic variation contributes to individual differences in susceptibility to respiratory infection.

title: Regulation of mammalian ciliary beating.
abstract: Recent advances in our understanding of the structure-function relationship of motile cilia with the 9 + 2 microtubular arrangement have helped explain some of the mechanisms of ciliary beat regulation by intracellular second messengers. These second messengers include cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) as well as calcium and pH. cAMP activates protein kinase A (PKA), which is localized to the axoneme. The cAMP-dependent phosphorylation of PKA's main target, originally described as p29 in Paramecium, seems to increase ciliary beat frequency (CBF) directly. The mechanism by which cGMP increases CBF is less well defined but involves protein kinase G and possibly PKA. Protein kinase C inhibits ciliary beating. The regulation mechanisms of CBF by calcium remain somewhat controversial, favoring an immediate, direct action of calcium on ciliary beating and a second cyclic nucleotide-dependent phase. Finally, intracellular pH likely affects CBF through direct influences on dynein arms.

title: Does nasal nitric oxide come from the sinuses?
abstract: The purpose of this study was to assess nitric oxide (NO) output by the nose and sinuses.



In one volunteer, the osteomeatal complex and sphenoethmoidal recess were occluded to isolate the nose from the sinuses. The antrum and frontal sinus were each punctured by two catheters and irrigated with air at constant flow. Nitric oxide output and its rate of accumulation in the absence of air flow were measured in each sinus and in the adjacent nasal cavity.



Prior to ostial occlusion, NO output in the nose was 96 nL/min. It decreased by 12% after blockage of all of the ostia. In the isolated sinuses, it was 190 nL/min (antrum) and 68 nL/min (frontal). After 5 minutes stagnation; NO concentration [NO] rose in the occluded sinuses to 24,700 nL/L in the antrum and 22,300 nL/L in the frontal sinus. In the nose, it increased to 29,000 nL/L. When the period of stagnation was prolonged in the frontal sinus, the [NO] reached a plateau. NO output and accumulation were not altered in the nose or either sinus by opening their ostia. In the antrum and frontal sinus, lidocaine reduced NO output and the rate of NO accumulation, but not in the nose.



In this volunteer, 88% of nasal NO was derived from the nose itself. Nitric oxide exchange between the frontal sinus, antrum, and nose was negligible. In the absence of air flow, [NO] rose to a plateau in the nose and frontal sinus. Lidocaine inhibited NO output in the sinuses but not the nose.

title: Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans.
abstract: The presence of nitric oxide (NO) in the exhaled air of humans and of anaesthetized rabbits and guinea pigs was demonstrated by chemiluminescence, diazotization and mass spectrometry. This NO is endogenously produced in the lung by an NO synthase, since its generation in guinea pigs and rabbits was inhibited by N omega-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine, inhibitors of this enzyme. The effect of the inhibitors was reversed by the precursor of NO synthesis, L-arginine. Since NO is produced by normal vascular endothelium for the physiological regulation of blood flow and pressure and also by activated macrophages to contribute to non-specific immunity, our experiments suggest that NO may play both vascular regulatory and host defence roles in pulmonary physiology and pathophysiology.

title: Nitric oxide in upper airways inflammatory diseases
abstract: In the human respiratory tract, the main production sites of exhaled nitric oxide (NO) are the nose and paranasal sinuses. In the upper airways, NO has been suggested to be involved at different levels with regulatory, protective, defensive or deleterious effects. Therefore, we review some aspects of the origin, metabolism, and functions of NO in the upper airways, together with the role of NO in some upper airways inflammatory diseases. Furthermore, we discuss the recent improvements in nasal NO measurements, which may be useful to better characterize the involvement of the NO produced by nose and paranasal sinuses in upper airways inflammatory diseases such as allergic rhinitis, nasal polyposis, sinusitis, primary ciliary dyskinesia, and cystic fibrosis.

title: Reduced nitric oxide in sinus epithelium of patients with radiologic maxillary sinusitis and sepsis.
abstract: Radiologic maxillary sinusitis is an important risk factor for development of bronchopneumonia in mechanically ventilated patients. Nitric oxide produced within the paranasal sinuses is considered to provide an antibacterial environment and to modulate mucociliary clearance function. We hypothesized that a reduced formation of nitric oxide might contribute to the compromised local host defense in radiologic maxillary sinusitis and measured nitric oxide levels directly within maxillary sinuses of septic patients with radiologic maxillary sinusitis (n = 11), whose sinuses were fenestrated to eliminate a possible septic focus. Data were compared with those of patients without airway inflammation (n = 11, control subjects). Despite local inflammation and infection, we found considerably lower maxillary nitric oxide levels than in control subjects (31 +/- 10 versus 2554 +/- 385 parts per billion, mean +/- standard error of the mean, p < 0.001). Consistently, immunohistochemical and in situ hybridization investigations revealed strongly reduced expression of inducible nitric oxide synthase. By applying ultrastructural immunolocalization, we identified cilia and microvilli of the maxillary sinus epithelium as the major nitric oxide production site in control subjects. Our findings provide evidence of markedly reduced nitric oxide production in maxillary sinuses of patients with radiologic maxillary sinusitis and sepsis, implicating impaired local host defense and an increased risk for secondary infections.

title: Expression of nitric oxide synthases in primary ciliary dyskinesia.
abstract: Nitric oxide is believed to play a central role in nonspecific defense of upper airways. Patients with primary ciliary dyskinesia have very low concentration of nasal nitric oxide, which may contribute to the chronic upper airway diseases encountered by these patients. The mechanisms underlying this drop of nasal nitric oxide in primary ciliary dyskinesia are still unknown. The goal of the present work was to study nitric oxide synthases expression in upper airway tissues from patients with primary ciliary dyskinesia. For this purpose, 5 patients with primary ciliary dyskinesia and 10 nonallergic age-matched patients without primary ciliary dyskinesia undergoing nasal polypectomy were included. Nasal nitric oxide concentration was measured before polypectomy, and nitric oxide synthase expression and function were studied in nasal polyps. The nasal nitric oxide in patients with primary ciliary dyskinesia was lower than that in patients without primary ciliary dyskinesia (13 [9-16] ppb versus 210 [167-254] ppb, P < .0001). Nitric oxide synthase 2 immunostaining was prominent at the apical part of the ciliated epithelial cells and was similar in both groups. Nitric oxide synthase 3 staining was restricted to endothelial cells in both groups. In addition, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity was superimposable to nitric oxide synthases 2 and 3 immunostaining, suggesting a preserved NADPH-activity of nitric oxide synthase. We therefore conclude that the drop in nasal nitric oxide in patients with primary ciliary dyskinesia is not secondary to the loss of nitric oxide synthase expression.

title: Iglewski and rhamnolipid biosynthesis genes . quorum-sensing systems in control of elastase Roles of Pseudomonas aeruginosa las and rhl
abstract: and rhamnolipid biosynthesis genes.

title: Quorum sensing: How bacteria can coordinate activity and synchronize their response to external signals?
abstract: Quorum sensing is used by a large variety of bacteria to regulate gene expression in a cell‐density‐dependent manner. Bacteria can synchronize population behavior using small molecules called autoinducers that are produced by cognate synthases and recognized by specific receptors. Quorum sensing plays critical roles in regulating diverse cellular functions in bacteria, including bioluminescence, virulence gene expression, biofilm formation, and antibiotic resistance. The best‐studied autoinducers are acyl homoserine lactone (AHL) molecules, which are the primary quorum sensing signals used by Gram‐negative bacteria. In this review we focus on the AHL‐dependent quorum sensing system and highlight recent progress on structural and mechanistic studies of AHL synthases and the corresponding receptors. Crystal structures of LuxI‐type AHL synthases provide insights into acyl‐substrate specificity, but the current knowledge is still greatly limited. Structural studies of AHL receptors have facilitated a more thorough understanding of signal perception and established the molecular framework for the development of quorum sensing inhibitors.

title: The Molecular Basis of Individual Differences in Phenylthiocarbamide and Propylthiouracil Bitterness Perception
abstract: Individual differences in perception are ubiquitous within the chemical senses: taste, smell, and chemical somesthesis . A hypothesis of this fact states that polymorphisms in human sensory receptor genes could alter perception by coding for functionally distinct receptor types . We have previously reported evidence that sequence variants in a presumptive bitter receptor gene (hTAS2R38) correlate with differences in bitterness recognition of phenylthiocarbamide (PTC) . Here, we map individual psychogenomic pathways for bitter taste by testing people with a variety of psychophysical tasks and linking their individual perceptions of the compounds PTC and propylthiouracil (PROP) to the in vitro responses of their TAS2R38 receptor variants. Functional expression studies demonstrate that five different haplotypes from the hTAS2R38 gene code for operatively distinct receptors. The responses of the three haplotypes we also tested in vivo correlate strongly with individuals' psychophysical bitter sensitivities to a family of compounds. These data provide a direct molecular link between heritable variability in bitter taste perception to functional variations of a single G protein coupled receptor that responds to compounds such as PTC and PROP that contain the N-C=S moiety. The molecular mechanisms of perceived bitterness variability have therapeutic implications, such as helping patients to consume beneficial bitter-tasting compounds-for example, pharmaceuticals and selected phytochemicals.

title: 3D Structure Prediction of TAS2R38 Bitter Receptors Bound to Agonists Phenylthiocarbamide (PTC) and 6-n-Propylthiouracil (PROP)
abstract: The G protein-coupled receptor (GPCR) TAS2R38 is a bitter taste receptor that can respond to bitter compounds such as phenylthiocarbamide (PTC) and 6-n-propylthiouracil (PROP). This receptor was chosen because its four haplotypes (based on three residue site polymorphism) hTAS2R38PAV, hTAS2R38AVI, hTAS2R38AAI, and hTAS2R38PVV are known to have dramatically different responses to PTC and PROP. We aimed to identify the protein-ligand interaction features that determine whether the bitter taste signal from this receptor is sent to the cortex. To do this we predicted the 3D structures of the TAS2R38 bitter taste receptor using our new BiHelix and SuperBiHelix Monte Carlo methods (No experimental determinations of the 3D structure have been reported for any taste receptors.). We find that residue 262 (2nd position in the polymorphism) is involved in the interhelical hydrogen bond network stabilizing the GPCR structure in tasters (hTAS2R38PAV, hTAS2R38AAI, and hTAS2R38PVV), while it is not in the nontaster (hTAS2R38AVI). This suggests that the hydrogen bond interactions between TM3 and TM6 or between TM5 and TM6 may play a role in activating this GPCR. To further validate these structures, we used the DarwinDock method to predict the binding sites and 3D structures for PTC and PROP bound to hTAS2R38PAV, hTAS2R38AVI, hTAS2R38AAI, and hTAS2R38PVV, respectively. Our results show that PTC and PROP can form H-bonds with the backbone of residue 262 in the tasters (hTAS2R38PAV, hTAS2R38AAI, and hTAS2R38PVV) but not in the nontaster (hTAS2R38AVI). Thus it appears that the hydrogen bond interaction between TM3 and TM6 may activate the receptor to pass the ligand binding signal to intracellular processes and that the H-bond between agonists and residue 262 in tasters is involved in the bitter tasting. This is in agreement with experimental observations, providing validation of the predicted ligand-protein complexes and also a potential activation mechanism for the TAS2R38 receptor.

title: Insights into the Binding of Phenyltiocarbamide (PTC) Agonist to Its Target Human TAS2R38 Bitter Receptor
abstract: Humans' bitter taste perception is mediated by the hTAS2R subfamily of the G protein-coupled membrane receptors (GPCRs). Structural information on these receptors is currently limited. Here we identify residues involved in the binding of phenylthiocarbamide (PTC) and in receptor activation in one of the most widely studied hTAS2Rs (hTAS2R38) by means of structural bioinformatics and molecular docking. The predictions are validated by site-directed mutagenesis experiments that involve specific residues located in the putative binding site and trans-membrane (TM) helices 6 and 7 putatively involved in receptor activation. Based on our measurements, we suggest that (i) residue N103 participates actively in PTC binding, in line with previous computational studies. (ii) W99, M100 and S259 contribute to define the size and shape of the binding cavity. (iii) W99 and M100, along with F255 and V296, play a key role for receptor activation, providing insights on bitter taste receptor activation not emerging from the previously reported computational models.

title: Modeling the human PTC bitter-taste receptor interactions with bitter tastants
abstract: AbstractWe employed the first principles computational method MembStruk and homology modeling techniques to predict the 3D structures of the human phenylthiocarbamide (PTC) taste receptor. This protein is a seven-transmembrane-domain G protein-coupled receptor that exists in two main forms worldwide, designated taster and nontaster, which differ from each other at three amino-acid positions. 3D models were generated with and without structural similarity comparison to bovine rhodopsin. We used computational tools (HierDock and ScanBindSite) to generate models of the receptor bound to PTC ligand to estimate binding sites and binding energies. In these models, PTC binds at a site distant from the variant amino acids, and PTC binding energy was equivalent for both the taster and nontaster forms of the protein. These models suggest that the inability of humans to taste PTC is due to a failure of G protein activation rather than decreased binding affinity of the receptor for PTC. Amino-acid substitutions in the sixth and seventh transmembrane domains of the nontaster form of the protein may produce increased steric hindrance between these two α-helices and reduce the motion of the sixth helix required for G protein activation.
 3D-model of phenylthiocarbamide (PTC) bound to PTC taste reception

title: Expression of taste receptors in Solitary Chemosensory Cells of rodent airways
abstract: BackgroundChemical irritation of airway mucosa elicits a variety of reflex responses such as coughing, apnea, and laryngeal closure. Inhaled irritants can activate either chemosensitive free nerve endings, laryngeal taste buds or solitary chemosensory cells (SCCs). The SCC population lies in the nasal respiratory epithelium, vomeronasal organ, and larynx, as well as deeper in the airway. The objective of this study is to map the distribution of SCCs within the airways and to determine the elements of the chemosensory transduction cascade expressed in these SCCs.MethodsWe utilized a combination of immunohistochemistry and molecular techniques (rtPCR and in situ hybridization) on rats and transgenic mice where the Tas1R3 or TRPM5 promoter drives expression of green fluorescent protein (GFP).ResultsEpithelial SCCs specialized for chemoreception are distributed throughout much of the respiratory tree of rodents. These cells express elements of the taste transduction cascade, including Tas1R and Tas2R receptor molecules, α-gustducin, PLCβ2 and TrpM5. The Tas2R bitter taste receptors are present throughout the entire respiratory tract. In contrast, the Tas1R sweet/umami taste receptors are expressed by numerous SCCs in the nasal cavity, but decrease in prevalence in the trachea, and are absent in the lower airways.ConclusionsElements of the taste transduction cascade including taste receptors are expressed by SCCs distributed throughout the airways. In the nasal cavity, SCCs, expressing Tas1R and Tas2R taste receptors, mediate detection of irritants and foreign substances which trigger trigeminally-mediated protective airway reflexes. Lower in the respiratory tract, similar chemosensory cells are not related to the trigeminal nerve but may still trigger local epithelial responses to irritants. In total, SCCs should be considered chemoreceptor cells that help in preventing damage to the respiratory tract caused by inhaled irritants and pathogens.

title: Cholinergic chemosensory cells in the trachea regulate breathing
abstract: In the epithelium of the lower airways, a cell type of unknown function has been termed “brush cell” because of a distinctive ultrastructural feature, an apical tuft of microvilli. Morphologically similar cells in the nose have been identified as solitary chemosensory cells responding to taste stimuli and triggering trigeminal reflexes. Here we show that brush cells of the mouse trachea express the receptors (Tas2R105, Tas2R108), the downstream signaling molecules (α-gustducin, phospholipase Cβ2) of bitter taste transduction, the synthesis and packaging machinery for acetylcholine, and are addressed by vagal sensory nerve fibers carrying nicotinic acetylcholine receptors. Tracheal application of an nAChR agonist caused a reduction in breathing frequency. Similarly, cycloheximide, a Tas2R108 agonist, evoked a drop in respiratory rate, being sensitive to nicotinic receptor blockade and epithelium removal. This identifies brush cells as cholinergic sensors of the chemical composition of the lower airway luminal microenvironment that are directly linked to the regulation of respiration.

title: Chemosensory brush cells of the trachea. A stable population in a dynamic epithelium.
abstract: Tracheal brush cells (BCs) are specialized epithelial chemosensors that use the canonical taste transduction cascade to detect irritants. To test whether BCs are replaced at the same rate as other cells in the surrounding epithelium of adult mice, we used 5-bromo-2'-deoxyuridine (BrdU) to label dividing cells. Although scattered BrdU-labeled epithelial cells are present 5-20 days after BrdU, no BCs are labeled. These data indicate that BCs comprise a relatively static population. To determine how BCs are generated during development, we injected 5-day-old mice with BrdU and found labeled BCs and non-BC epithelial cells 5 days after BrdU. During the next 60 days, the percentage of labeled BCs increased, whereas the percentage of other labeled cell types decreased. These data suggest that BCs are generated from non-BC progenitor cells during postnatal tracheal growth. To test whether the adult epithelium retains the capacity to generate BCs, tracheal epithelial cells were recovered from adult mice and grown in an air-liquid interface (ALI) culture. After transition to differentiation conditions, BCs are detected, and comprise 1% of the total cell population by Day 14. BrdU added to cultures before the differentiation of BCs was chased into BCs, indicating that the increase in BC density is attributable to the proliferation of a non-BC progenitor. We conclude that: (1) BCs are normally a static population in adult mice; (2) BC progenitors proliferate and differentiate during neonatal development; and (3) BCs can be regenerated from a proliferative population resident in adult epithelium.

title: A new fate for old cells: brush cells and related elements
abstract: Over the past 50 years, hundreds of studies have described those cells that are characterized by a brush of rigid apical microvilli with long rootlets, and which are found in the digestive and respiratory apparatuses. These cells have been given names such as brush cells, tuft cells, fibrillovesicular cells, multivesicular cells and caveolated cells. More recently, it has been realized that all these elements may represent a single cell type, probably with a chemosensory role, even if other functions (e.g. secretory or absorptive) seem to be possible. Very recent developments have permitted a partial definition of the chemical code characterizing these elements, revealing the presence of molecules involved in chemoreceptorial cell signalling. A molecular cascade, similar to those characterizing the gustatory epithelium, seems to be present in these elements. These new data suggest that these elements can be considered solitary chemosensory cells with the presence of the apical ‘brush’ as an inconsistent feature. They seem to comprise a diffuse chemosensory system that covers large areas (probably the whole digestive and respiratory apparatuses) with analogies to chemosensory systems described in aquatic vertebrates.

title: Cholinergic brush cells in the trachea mediate respiratory responses to quorum sensing molecules.
abstract: The airway epithelial surface is constantly exposed to inhaled environmental factors and pathogens. Bitter "tasting" bacterial products such as quorum sensing molecules (QSM) can be detected by solitary chemosensory cells of the upper respiratory tract. Recently, we have shown that tracheal brush cells are cholinergic chemosensory cells affecting the respiration upon stimulation with bitter substances. Here, we explore the hypothesis that tracheal brush cells are capable of detection of bacterial products such as QSM resulting in changes in respiration and in induction of local effects, e.g. regulation of mucociliary clearance.



Functional analyses of respiration were performed in the trachea using a newly established model for investigation of respiration in spontaneously breathing anesthetized mice upon isolated tracheal stimulation. Influence of N-3-oxododecanoyl-homoserine lactone (3-OxoC(12)-HSL) on cilia-driven particle transport speed (PTS) in the airways was investigated in acutely excised and submerged mouse tracheae.



3-OxoC(12)-HSL, a Pseudomonas aeruginosa quorum sensing autoinducer, caused a drop in the respiratory rate 2 min after the application at the mucosal surface. The 3-OxoC(12)-HSL-induced effect on respiration was abolished by inhibition of nicotinic receptors with mecamylamine and by removal of the respiratory epithelium. At the same concentration, 3-OxoC(12)-HSL enhanced significantly PTS on the mucosal surface.



We conclude that cholinergic airway epithelial cells sense bacterial QSM in the airway lining fluid and communicate this to the CNS via ACh release and nicotinic stimulation of sensory neurons. In addition, QSM enhance PTS.

title: TAS2R activation promotes airway smooth muscle relaxation despite β(2)-adrenergic receptor tachyphylaxis.
abstract: Recently, bitter taste receptors (TAS2Rs) were found in the lung and act to relax airway smooth muscle (ASM) via intracellular Ca(2+) concentration signaling generated from restricted phospholipase C activation. As potential therapy, TAS2R agonists could be add-on treatment when patients fail to achieve adequate bronchodilation with chronic β-agonists. The β(2)-adrenergic receptor (β(2)AR) of ASM undergoes extensive functional desensitization. It remains unknown whether this desensitization affects TAS2R function, by cross talk at the receptors or distal common components in the relaxation machinery. We studied intracellular signaling and cell mechanics using isolated human ASM, mouse tracheal responses, and human bronchial responses to characterize TAS2R relaxation in the context of β(2)AR desensitization. In isolated human ASM, magnetic twisting cytometry revealed >90% loss of isoproterenol-promoted decrease in cell stiffness after 18-h exposure to albuterol. Under these same conditions of β(2)AR desensitization, the TAS2R agonist chloroquine relaxation response was unaffected. TAS2R-mediated stimulation of intracellular Ca(2+) concentration in human ASM was unaltered by albuterol pretreatment, in contrast to cAMP signaling, which was desensitized by >90%. In mouse trachea, β(2)AR desensitization by β-agonist amounted to 92 ± 6.0% (P < 0.001), while, under these same conditions, TAS2R desensitization was not significant (11 ± 3.5%). In human lung slices, chronic β-agonist exposure culminated in 64 ± 5.7% (P < 0.001) desensitization of β(2)AR-mediated dilation of carbachol-constricted airways that was reversed by chloroquine. We conclude that there is no evidence for physiologically relevant cross-desensitization of TAS2R-mediated ASM relaxation from chronic β-agonist treatment. These findings portend a favorable therapeutic profile for TAS2R agonists for the treatment of bronchospasm in asthma or chronic obstructive lung disease.

title: Agonist-promoted homologous desensitization of human airway smooth muscle bitter taste receptors.
abstract: Bitter taste receptors (TAS2Rs) were shown to be expressed in human airway smooth muscle (ASM). They couple to specialized [Ca(2+)](i) release, leading to membrane hyperpolarization, the relaxation of ASM, and marked bronchodilation. TAS2Rs are G-protein-coupled receptors, known to undergo rapid agonist-promoted desensitization that can limit therapeutic efficacy. Because TAS2Rs represent a new drug target for treating obstructive lung disease, we investigated their capacity for rapid desensitization, and assessed their potential mechanisms. The pretreatment of human ASM cells with the prototypic TAS2R agonist quinine resulted in a 31% ± 5.1% desensitization of the [Ca(2+)](i) response from a subsequent exposure to quinine. No significant change in the endothelin-stimulated [Ca(2+)](i) response was attributed to the short-term use of quinine, indicating a homologous form of desensitization. The TAS2R agonist saccharin also evoked desensitization, and cross-compound desensitization with quinine was evident. Desensitization of the [Ca(2+)](i) response was attenuated by a dynamin inhibitor, suggesting that receptor internalization (a G-protein coupled receptor kinase [GRK]-mediated, β-arrestin-mediated process) plays an integral role in the desensitization of TAS2R. Desensitization was insensitive to antagonists of the second messenger kinases protein kinase A and protein kinase C. Using intact airways, short-term, agonist-promoted TAS2R desensitization of the relaxation response was also observed. Thus these receptors, which represent a potential novel target for direct bronchodilators, undergo a modest degree of agonist-promoted desensitization that may affect clinical efficacy. Collectively, the results of these mechanistic studies, along with the multiple serines and threonines in intracellular loop 3 and the cytoplasmic tail of TAS2Rs, suggest a GRK-mediated mode of desensitization.

title: The bitter taste receptor (TAS2R) agonists denatonium and chloroquine display distinct patterns of relaxation of the guinea pig trachea.
abstract: Activation of taste receptors (TAS2Rs) by bitter taste agonists has been reported to cause bronchodilation. The aim of this study was to extend the information on the effects of bitter taste agonists on responses induced by different contractile mediators in a standard airway physiology preparation. Isometric responses were assessed in guinea pig trachea (GPT). TAS2R agonists were administered either to segments precontracted with different agonists for contraction or given before challenge with the different contractile stimuli, including antigen in tissues from ovalbumin-sensitized animals. TAS2R mRNA expression on GPT epithelium and smooth muscle was measured with real-time PCR. Denatonium, chloroquine, thiamine, and noscapine induced concentration-dependent relaxations (R(max): 98.3 ± 1.6, 100.0 ± 0.0, 100.0 ± 0.0, and 52.3 ± 1.1% of maximum, respectively, in the presence of indomethacin) in segments precontracted with carbachol. The receptors for denatonium (TAS2R4, TAS2R10) and chloroquine (TAS2R3, TAS2R10) were expressed in GPT. Whereas denatonium selectively inhibited contractions induced by carbachol, chloroquine uniformly inhibited contractions evoked by prostaglandin E(2), the thromboxane receptor agonist U-46619, leukotriene D(4), histamine, and antigen. The effects of denatonium, but not those of chloroquine, were partly inhibited by blockers of the large Ca(2+)-activated K(+) channels and decreased by an increase of the level of precontraction. In conclusion, TAS2R agonists mediated strong relaxations and substantial inhibition of contractions in GPT. Chloroquine and denatonium had distinct patterns of activity, indicating different signaling mechanisms. The findings reinforce the hypothesis that TAS2Rs are potential targets for the development of a new class of more efficacious agonists for bronchodilation.

title: The expression and relaxant effect of bitter taste receptors in human bronchi
abstract: BackgroundBitter-taste receptors (TAS2Rs) have recently been involved in the relaxation of mouse and guinea pig airways, and increased expression of TAS2Rs was shown in blood leucocytes from asthmatic children. We sought to identify and characterize the TAS2Rs expressed in isolated human bronchi and the subtypes involved in relaxation.MethodsHuman bronchi were isolated from resected lungs and TAS2R transcripts were assessed with RT-qPCR. Relaxation to TAS2R agonists was tested in organ bath in the presence or absence of pharmacological modulators of the signalling pathways involved in bronchial relaxation.ResultsWe detected the expression of TAS2R transcripts in human bronchi. The non-selective agonists chloroquine, quinine, caffeine, strychnine and diphenidol produced a bronchial relaxation as effective and potent as theophylline but much less potent than formoterol and isoproterenol. Denatonium, saccharin and colchicine did not produce relaxation. Receptor expression analysis together with the use of selective agonists suggest a predominant role for TAS2R5, 10 and 14 in bitter taste agonist-induced relaxation. The mechanism of relaxation was independent of the signalling pathways modulated by conventional bronchodilators and may be partly explained by the inhibition of phosphatidylinositol-3-kinases. TAS2Rs may constitute a new therapeutic target in chronic obstructive lung diseases such as asthma.

title: Bitter taste receptor function in asthmatic and nonasthmatic human airway smooth muscle cells.
abstract: Bitter taste receptors (TAS2Rs) have recently been found to be expressed on human airway smooth muscle (HASM), and their activation results in marked relaxation. These agents have been proposed as a new class of bronchodilators in the treatment of obstructive lung diseases because they act via a different mechanism than β-agonists. The TAS2R signal transduction pathway in HASM has multiple elements that are potentially subject to regulation by inflammatory, genetic, and epigenetic mechanisms associated with asthma. To address this, expression, signaling, and physiologic functions of the three major TAS2Rs (subtypes 10, 14, and 31) on HASM were studied. Transcript expression of these TAS2Rs was not decreased in HASM cells derived from donors with asthma compared with those without asthma (n = 6 from each group). In addition, intracellular calcium ([Ca(2+)]i) signaling using TAS2R subtype-specific agonists (diphenhydramine, chloroquine, saccharin, and flufenamic acid) was not impaired in the cells derived from donors with asthma, nor was the response to quinine, which activates all three subtypes. HASM cell mechanics measured by magnetic twisting cytometry revealed equivalent TAS2R-mediated relaxation of methacholine-treated cells between the two groups. Human precision-cut lung slices treated with IL-13 caused a decrease in β-agonist (formoterol)-mediated relaxation of carbachol-contracted airways compared with control slices. In contrast, TAS2R-mediated relaxation was unaffected by IL-13. We conclude that TAS2R expression or function is unaffected in HASM cells derived from patients with asthma or the IL-13 inflammatory environment.