Biology subjects

Palmer, J. N.

Publications and source records attributed to Palmer, J. N..

16 recordsLinked to original sources

bioRxiv2026

Targeting cyclic-AMP/phosphodiesterase signaling to quiet nasal tuft cell calcium responses

Background: Airway tuft cells, also called solitary chemosensory cells, are enriched in nasal polyps and are likely the main epithelial source of interleukin-25 (IL-25) in chronic rhinosinusitis with nasal polyps (CRSwNP). Tuft cells are activated through G protein-coupled receptors (GPCRs) that signal through Ca2+. These receptors may also lower cyclic AMP (cAMP), but whether the cAMP pathway can be used to suppress tuft cell responses is unknown. Methods: Ca2+ and cAMP were measured by live cell imaging in isolated Trpm5-GFP mouse nasal tuft cells, and Ca2+ responses were measured in primary human nasal epithelial air-liquid interface (ALI) cultures from patients with CRSwNP. Defensin and IL-25 release were measured by ELISA and ciliary beat frequency (CBF) by high-speed imaging. Phosphodiesterase (PDE) expression was assessed using publicly available single cell RNA sequencing datasets. Results: Agonists of bitter taste receptors (T2Rs), the succinate receptor SUCNR1, and the A1 adenosine receptor increased Ca2+ in GFP-positive mouse nasal tuft cells. T2R and A1 receptor stimulation also lowered cAMP, and these decreases were blocked by pertussis toxin. Glucose, which activates taste family 1 receptors, increased cAMP. Tuft cells were enriched for Pde2a. PDE2A-selective inhibitors (PF-05180999 and BAY 60-7550) increased cAMP and reduced agonist-evoked Ca2+ responses in mouse tuft cells. In human nasal ALIs, both compounds reduced denatonium-evoked Ca2+ responses, defensin release, and IL-25 release without increasing CBF. Conclusions: GPCRs that activate nasal tuft cells also lower cAMP, and by raising cAMP through PDE2A inhibition, tuft cell Ca2+ signaling and IL-25 release were reduced in vitro.

physiology↗

bioRxiv2026

MS4A8B regulates Orai1-dependent Ca2+ influx to control motile cilia function in human nasal epithelial cells

Motile cilia demonstrate coordinated beating to propel fluids across epithelial tissues, and changes to their beating frequency are largely regulated by intracellular second messengers including Ca2+. In the airway epithelium, ciliary beating is essential to mucociliary clearance. Mucociliary clearance involves trapping inhaled pathogens and irritants in sticky mucus lining the airways for motile cilia to sweep away contaminated mucus, preventing infection and reducing general airway inflammation. Many chronic respiratory diseases, including chronic rhinosinusitis and asthma, are characterized by an acquired ciliary dysfunction. Despite the importance of Ca2+ signaling in cilia physiology, the identity and molecular mechanisms governing localized ciliary Ca2+ transport remain poorly understood. MS4A8B is an uncharacterized cilia-localized transmembrane protein. Other MS4A homologs have been indirectly linked to Ca2+ signaling via uncharacterized mechanisms. Using primary human nasal epithelial cells differentiated at air-liquid interface, we demonstrated that MS4A8B regulates motile cilia function. MS4A8B knockdown impairs ciliary beating and impacts cilia structure. Live-cell imaging combined with genetic analysis revealed that MS4A8B potentiates Orai1-mediated Ca2+ influx. Co-immunoprecipitation and FRET microscopy in ectopic expression systems demonstrated that MS4A8B interacts with Orai1 channels. Orai1 was further identified to reside in motile cilia of primary human nasal epithelial cells, allowing ciliary beat frequency to be stimulated by Orai1 agonists including arachidonic acid. MS4A8B functional coupling with Orai1 acts as an autonomous cilia signaling network. Targeting this compartmentalized signaling pathway offers a novel therapeutic approach to restore or enhance mucociliary clearance in airway diseases.

physiology↗

bioRxiv2025

Human olfactory organoids as an in vitro model of the olfactory epithelium

Abstract/Key PointsO_LIA novel human olfactory epithelial organoid model has been generated from primary cell culture. C_LIO_LIHuman olfactory organoids exhibit immunostaining and gene expression for olfactory cells. C_LIO_LIOlfactory organoids mobilize Ca2+ in response to odorants, a surrogate for neuronal function. C_LI

cell biology↗

bioRxiv2024

Effects of Pseudomonas aeruginosa pyocyanin and 1-hydroxyphenazine on intracellular calcium, mitochondrial function, and viability in human nasal epithelial cells

Pseudomonas aeruginosa is an opportunistic pathogen that produces phenazine metabolites pyocyanin and 1-hydroxyphenazine that have been suggested to have detrimental effects on mitochondrial function and reactive oxygen species (ROS) production. Prior studies have suggested activation of Ca2+ signaling by pyocyanin in an airway cell line, while others have shown apoptotic effects on cancer cells. Ca2+ is tightly linked to both normal mitochondrial function as well as mitochondrial ROS and apoptosis during mitochondrial Ca2+ overload. We found that pyocyanin but not 1-hydroxyphenazine induced both cytosolic and mitochondrial Ca2+ increases in RPMI 2650 and primary human nasal epithelial cells (HNEC). Similar results were seen in HNEC air-liquid interface (ALI) cells, but these cells did not display a cytosolic Ca2+ response after treatment with pyocyanin. In RPMI 2650, activation of PKC, ER Ca2+ release, and PLC inhibition, indicated potential GPCR activation from pyocyanin. Similarly, ROS production increased after treatment with pyocyanin, but not 1-hydroxyphenazine in all 3 cell types, but with stark differences in CF and non-CF ALIs. In HNEC ALI, pyocyanin reduced ciliary beat frequency (CBF) after 4 hours, while 1-hydroxpyhenazine did not. Despite this, both pyocyanin and 1-hydroxyphenazine decreased in cell viability in RPMI 2650 nasal carcinoma cells but not in HNEC at 24 hours. However, in both RPMI 2650 and HNEC, mitochondrial membrane potential acutely decreased after treatment with either pyocyanin or 1-hydroxyphenazine. Finally, 24-hour pyocyanin treatment decreased expression of ER stress genes in some cancer cells, but not in non-cancerous HNEC. Our data suggest that Ca2+ signaling is not required for acute effects of 1-hydroxyphenazine or pyocyanin on mitochondrial function. The greater sensitivity of RPMI 2650 cells to pyocyanin-induced and 1-hydroxyphenzine-induced cytotoxicity compared with primary cells suggests that these compounds might have some applicability in treating nasal squamous carcinomas or other types of head and neck squamous carcinomas. Although the exact mechanisms of pyocyanin induced apoptosis remains uncertain, the downregulation of the ER stress response may play a role.

physiology↗

bioRxiv2022

Savory Signaling: T1R umami receptor modulates endoplasmic reticulum calcium store content and release dynamics in airway epithelial cells

T1Rs are expressed in solitary chemosensory cells of the upper airway where they detect apical glucose levels and repress bitter taste receptor Ca2+ signaling pathways. Microbial growth leads to a decrease in apical glucose levels. T1Rs detect this change and liberate bitter taste receptor signaling, initiating an innate immune response to both kill and expel pathogens through releasing antimicrobial peptides and increasing nitric oxide production and ciliary beat frequency. However, chronic inflammation due to disease, smoking, or viral infections causes a remodeling of the airway epithelial. The resulting squamous metaplasia causes a loss of multi-ciliated cells and solitary chemosensory cells, replaced by basal epithelial cells. To understand how T1R function is altered during disease, we used basal epithelial cells as a model to study the function of T1R3 on Ca2+ signaling dynamics. We found that both T1R1 and T1R3 detect amino acids and signal via cAMP, increasing the responsiveness of the cells to Ca2+ signaling stimuli. Either knocking down T1R1/3 or treating wild-type cells with MEM amino acids caused a reduction in ER Ca2+ content through a non-cAMP signaled pathway. Treatment with amino acids led to a reduction in downstream denatonium-induced Ca2+-signaled caspase activity. Thus, amino acids may be used to reduce unwanted apoptosis signaling in treatments containing bitter compounds.

physiology↗

bioRxiv2022

Akt activator SC79 stimulates antibacterial nitric oxide production from human nasal epithelial cells and increases macrophage phagocytosis in vitro

BackgroundThe role of the Akt serine/threonine kinase family in airway innate immunity is relatively unstudied compared with other pathways. Akt can phosphorylate and activate the endothelial nitric oxide synthase (eNOS) isoform expressed in airway epithelial ciliated cells. NO production by nasal epithelial cells often has antibacterial and antiviral effects. Increasing nasal epithelial NO production may be a useful anti-pathogen strategy for respiratory infections in diseases like chronic rhinosinusitis. We hypothesized that a small molecule Akt activator, SC79, might induce nasal epithelial cell NO production with bactericidal effects. MethodsWe tested the antibacterial-stimulatory effects of SC79 in primary nasal epithelial cells isolated from residual surgical material and grown at air-liquid interface. Because macrophages also use NO signaling to enhance phagocytosis, we also tested effects of SC79 in human macrophages differentiated from monocytes obtained from healthy apheresis donors. ResultsLive cell imaging of an NO-sensitive fluorescent dye revealed that SC79 induced dose-dependent NO production. Pharmacology and genetic knockdown revealed that this NO production is dependent on eNOS and Akt. The NO released into the airway surface liquid was sufficient to kill both lab and clinical strains of P. aeruginosa in a co-culture bacterial killing assay. SC79 enhanced bacterial phagocytosis in a NO-dependent and Akt-dependent manner. No overt toxicity (LDH release) or inflammatory effects (IL8 transcription) were observed in nasal cells or macrophages over 24 hrs. ConclusionsTogether, these data suggest that multiple innate immune pathways might be stimulated by SC79 delivered via topical nasal rinse or spray. Activating Akt using SC79 or another compound might have beneficial antipathogen effects in respiratory infections.

physiology↗

bioRxiv2022

Taste receptor T1R3 in nasal cilia detects Staphylococcus aureus D-amino acids to enhance apical glucose uptake

Bitter (T2R) and sweet/umami (T1R) taste receptors serve chemosensory roles throughout the body. In airway cilia, T2Rs detect bacterial metabolites to stimulate bactericidal nitric oxide. T1Rs in solitary chemosensory cells detect glucose in airway surface liquid (ASL) and bacterial D-stereoisomer amino acids to regulate antimicrobial peptides. Using differentiated air-liquid interface cultures of primary nasal cells, we show that the T1R3 receptor is also expressed in human and mouse nasal cell cilia. D-amino acids produced at low mM concentrations by Staphylococcus aureus activate T1R3 to decrease ASL glucose and increase apical glucose uptake by increasing GLUT2 and GLUT10 expression through a {beta}-arrestin pathway. Our data suggests that T1R3 localized to cilia functions as an immune detector for D-amino acids to reduce ASL glucose and potentially limit bacterial growth. Given that glucose protected S. aureus against bactericidal NO produced during T2R activation, the reduced ASL glucose with T1R3 activation may also sensitize bacteria to other innate defenses. HIGHLIGHTSO_LINasal motile cilia express the T1R3 subunit of the sweet taste receptor C_LIO_LIS. aureus D-amino acids activate cilia T1R3 to enhance mucosal glucose uptake C_LIO_LIReduced airway surface liquid glucose likely reduces S. aureus growth C_LIO_LIReduced airway glucose also sensitizes S. aureus to epithelial NO production C_LI

physiology↗

bioRxiv2022

Bitter taste receptor-stimulated nitric oxide innate immune responses are reduced by loss of CFTR function in nasal epithelial cells and macrophages

Bitter taste receptors (T2Rs) are G protein-coupled receptors identified on the tongue but expressed all over the body, including in airway cilia and macrophages, where T2Rs serve an immune role. T2R isoforms detect bitter metabolites (quinolones and acyl-homoserine lactones) secreted by gram negative bacteria, including Pseudomonas aeruginosa, a major pathogen in cystic fibrosis (CF). T2R activation by bitter bacterial products triggers calcium-dependent nitric oxide (NO) production. In airway cells, the NO increases mucociliary clearance and has direct antibacterial properties. In macrophages, the same pathway enhances phagocytosis. Because prior studies linked CF with reduced NO, we hypothesized that CF cells may have reduced T2R/NO responses. Using qPCR, immunofluorescence, and live cell imaging of air-liquid interface cultures, we found primary nasal epithelial cells from both CF and non-CF patients exhibited similar T2R expression, localization, and calcium signals. However, CF cells exhibited reduced NO production also observed in immortalized CFBE41o-CF cells and non-CF 16HBE cells CRISPR modified with CF-causing mutations in the CF transmembrane conductance regulator (CFTR). NO was restored by VX-770/VX-809 corrector/potentiator pre-treatment, suggesting reduced NO in CF cells is due to loss of CFTR function. In nasal cells, reduced NO correlated with reduced ciliary and antibacterial responses. In primary human macrophages, inhibition of CFTR reduced NO production and phagocytosis during T2R stimulation. Together, these data suggest an intrinsic deficiency in T2R/NO signaling caused by loss of CFTR function that may contribute to intrinsic susceptibilities of CF patients to P. aeruginosa and other gram-negative bacteria that activate T2Rs.

physiology↗

bioRxiv2022

Cilia stimulatory and antibacterial activities of bitter receptor agonist diphenhydramine: insights into potential complimentary strategies for CF nasal infections

BACKGROUNDBitter compounds increase ciliary beating and nitric oxide (NO) production in nasal epithelial cells through T2Rs in motile cilia. We examined expression of cilia T2Rs and both host and bacterial responses to T2R14 agonist diphenhydramine. METHODUsing cultured human nasal epithelial cells grown at air liquid interface, we measured expression of T2Rs via qPCR. We measured effects of diphenhydramine on ciliary beat frequency via high-speed imaging and nitric oxide production via fluorescent dye DAF-FM. We measured effects of diphenhydramine on growth of lab and clinical strains of Pseudomonas aeruginosa. We measured biofilm formation of P. aeruginosa using crystal violet staining and surface attachment of P. aeruginosa to cystic fibrosis bronchial epithelial (CBFE41o-) cells using CFU counting. RESULTST2R expression increased with mucocilliary differentiation and did not vary between CF and non-CF ALIs. Treatment with P. aeruginosa flagellin decreased expression of diphenhydramine-responsive T2R14 and 40, among other isoforms. Diphenhydramine increased both NO and CBF. Increases in CBF were disrupted after flagellin treatment. Diphenhydramine impaired growth, biofilm production, and surface attachment of P. aeruginosa. CONCLUSIONST2R expression is similar between normal and CF cells but decreases with flagellin treatment. Utilizing T2R agonists as therapeutics within the context of CF, P. aeruginosa infections may require co-treatment with anti-inflammatories to prevent the reduction of T2R expression with TLR activation. T2R agonist diphenhydramine increases NO production and CBF while also decreasing bacterial growth and biofilm production, and thus diphenhydramine or derivate compounds may have potential clinical usefulness in CF infections as a topical therapy. HIGHLIGHTSO_LIT2R14 agonist diphenhydramine increases nitric oxide production and cilia beating C_LIO_LIFlagellin decreases T2R14 expression in primary airway epithelial cells C_LIO_LIT2R14 agonist Diphenhydramine inhibits Pseudomonas growth and biofilm formation C_LI

physiology↗

bioRxiv2021

HSP90 function is required for T2R bitter taste receptor nitric oxide production and innate immune responses in human airway epithelial cells and macrophages

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.

physiology↗

bioRxiv2021

The bitter end: T2R bitter receptor agonists elevate nuclear calcium and induce apoptosis in non-ciliated airway epithelial cells

Bitter taste receptors (T2Rs) localize to airway motile cilia and initiate innate immune responses in retaliation to bacterial quorum sensing molecules (acyl-homoserine lactones and quinolones). Activation of T2Rs leads to calcium-driven NO production that increases cilia beating and directly kills bacteria. Several airway diseases, including chronic rhinosinusitis, COPD, and cystic fibrosis, are characterized by epithelial remodeling, including loss of motile cilia and/or squamous metaplasia. To understand the function of T2Rs within the altered landscape of airway disease, we studied T2Rs in non-ciliated airway cell lines and primary cells de-differentiated to a squamous phenotype. In differentiated cells, T2Rs localize to cilia, however in de-differentiated, non-ciliated cells they localize to the nucleus. Cilia and nuclear import utilize many shared proteins, thus in the absence of motile cilia some T2Rs may target to the nucleus. T2R agonists selectively elevated both nuclear and mitochondrial calcium through a G-protein-coupled receptor, phospholipase C, and InsP3 receptor-dependent mechanism. Additionally, T2R agonists decreased nuclear cAMP, increased nitric oxide, and increased cGMP, consistent with T2R signaling. Furthermore, exposure to T2R agonists led to nuclear calcium-induced mitochondrial depolarization and caspase activation. T2R agonists induced apoptosis in primary bronchial and nasal cells differentiated at air-liquid interface but then induced to a squamous phenotype by apical submersion. Air-exposed well-differentiated cells did not die. This T2R-induced apoptosis may be a last-resort defense against infection, possibly when bacteria have breached the epithelial barrier and reach non-ciliated cells below. However, it may also increase susceptibility of de-differentiated or remodeled epithelia to damage by bacterial metabolites. Moreover, the T2R-activated apoptosis pathway occurs in airway cancer cells. T2Rs may thus contribute to microbiome-tumor cell crosstalk in airway cancers. T2R agonists may also be useful topical therapeutics (e.g., delivered by nasal rinse or nebulizer) for activating airway cancer cell apoptosis without killing surrounding differentiated tissue.

physiology↗

bioRxiv2020

SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial derived cells and cardiomyocytes

Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection, induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung, and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, while PKR activation is evident in iAT2 and iCM. In SARS-CoV-2 infected Calu-3 and A549ACE2 lung-derived cell lines, IFN induction remains relatively weak; however activation of OAS-RNase L and PKR is observed. This is in contrast to MERS-CoV, which effectively inhibits IFN signaling as well as OAS-RNase L and PKR pathways, but similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, both OAS-RNase L and PKR are activated in MAVS knockout A549ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2. SignificanceSARS-CoV-2 emergence in late 2019 led to the COVID-19 pandemic that has had devastating effects on human health and the economy. Early innate immune responses are essential for protection against virus invasion. While inadequate innate immune responses are associated with severe COVID-19 diseases, understanding of the interaction of SARS-CoV-2 with host antiviral pathways is minimal. We have characterized the innate immune response to SARS-CoV-2 infections in relevant respiratory tract derived cells and cardiomyocytes and found that SARS-CoV-2 activates two antiviral pathways, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR), while inducing minimal levels of interferon. This in contrast to MERS-CoV which inhibits all three pathways. Activation of these pathways may contribute to the distinctive pathogenesis of SARS-CoV-2.

microbiology↗

bioRxiv2020

Altered polarization of PAR-2 signaling during airway epithelial remodeling

BackgroundProtease-activated receptor 2 (PAR-2) is activated by proteases involved in allergy and triggers airway epithelial secretion and inflammation. PAR-2 is normally expressed basolaterally in differentiated nasal ciliated cells. ObjectiveWe tested if epithelial remodeling during diseases characterized by loss of cilia and squamous metaplasia may alter PAR-2 polarization. MethodsEndogenous PAR-2 responses were measured by live cell calcium and cilia imaging, measurement of fluid secretion, and quantification of cytokines. We utilized airway squamous cell lines, primary differentiated air-liquid interface cultures, and tissue explants. Cells were exposed to disease-related modifiers that alter epithelial morphology, including IL-13, cigarette smoke condensate, and retinoic acid deficiency. We used concentrations and exposure times that altered epithelial morphology without causing breakdown of the epithelial barrier, likely reflecting early disease states. ResultsPAR-2 signaling in airway squamous cells activated calcium and inflammatory responses. Squamous cells cultured at air liquid interface (ALI) responded to PAR-2 agonists applied both apically and basolaterally. Primary well-differentiated nasal epithelial ALI cultures responded only to basolateral PAR-2 stimulation. Primary cultures exposed to IL-13, cigarette smoke condensate, or reduced retinoic acid responded to both apical and basolateral PAR-2 stimulation. Nasal polyp tissue, but not control middle turbinate, exhibited apical calcium responses to PAR-2 stimulation. However, isolated ciliated cells from both polyp and turbinate maintained basolateral PAR-2 polarization. ConclusionsSquamous metaplasia and/or loss of cilia enhances apical PAR-2 responses. Altered PAR-2 polarization in dedifferentiated or remodeled epithelia may contribute to increased sensitivity to inhaled protease allergens in inflammatory airway diseases.

physiology↗

bioRxiv2019

Inverse regulation of secretion and inflammation in human airway gland serous cells by neuropeptides upregulated in allergy and asthma

Airway submucosal gland serous cells are sites of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) and are important for fluid secretion in conducting airways from the nose down to small bronchi. We tested if serous cells from human nasal turbinate glands secrete bicarbonate (HCO3-), important for mucus polymerization, during stimulation with the cAMP-elevating agonist vasoactive intestinal peptide (VIP) and if this requires CFTR. Isoalted serous cells stimulated with VIP exhibited a ~20% cAMP-dependent decrease in cell volume and a ~0.15 unit decrease in intracellular pH (pHi), reflecting activation of Cl- and HCO3- secretion, respectively. Pharmacology, ion substitution, and studies using cells from CF patients suggest serous cell HCO3- secretion is mediated by conductive efflux directly through CFTR. Interestingly, we found that neuropeptide Y (NPY) reduced VIP-evoked secretion by blunting cAMP increases and reducing CFTR activation through Gi-coupled NPY1R. Culture of primary gland serous cells in a model that maintained a serous phenotype confirmed the activating and inhibiting effects of VIP and NPY, respectively, on fluid and HCO3- secretion. Moreover, VIP enhanced secretion of antimicrobial peptides and antimicrobial efficacy of gland secretions while NPY reduced antimicrobial secretions. In contrast, NPY enhanced the release of cytokines during inflammatory stimuli while VIP reduced cytokine release through a mechanism requiring CFTR conductance. As levels of VIP and NPY are up-regulated in disease like allergy, asthma, and chronic rhinosinusitis, the balance of these two peptides in the airway may control airway mucus rheology and inflammatory responses through gland serous cells.

physiology↗

bioRxiv2018

Species-level bacterial community profiling of the healthy sinonasal microbiome using Pacific Biosciences sequencing of full-length 16S rRNA genes

BackgroundPan-bacterial 16S rRNA microbiome surveys performed with massively parallel DNA sequencing technologies have transformed community microbiological studies. Current 16S profiling methods, however, fail to provide sufficient taxonomic resolution and accuracy to adequately perform species-level associative studies for specific conditions. This is due to the amplification and sequencing of only short 16S rRNA gene regions, typically providing for only family- or genus-level taxonomy. Moreover, sequencing errors often inflate the number of taxa present. Pacific Biosciences (PacBios) long-read technology in particular suffers from high error rates per base. Herein we present a microbiome analysis pipeline that takes advantage of PacBio circular consensus sequencing (CCS) technology to sequence and error correct full-length bacterial 16S rRNA genes, which provides high-fidelity species-level microbiome data\n\nResultsAnalysis of a mock community with 20 bacterial species demonstrated 100% specificity and sensitivity. Examination of a 250-plus species mock community demonstrated correct species-level classification of >90% of taxa and relative abundances were accurately captured. The majority of the remaining taxa were demonstrated to be multiply, incorrectly, or incompletely classified. Using this methodology, we examined the microgeographic variation present among the microbiomes of six sinonasal sites, by both swab and biopsy, from the anterior nasal cavity to the sphenoid sinus from 12 subjects undergoing trans-sphenoidal hypophysectomy. We found greater variation among subjects than among sites within a subject, although significant within-individual differences were also observed. Propiniobacterium acnes (recently renamed Cutibacterium acnes [1]) was the predominant species throughout, but was found at distinct relative abundances by site.\n\nConclusionsOur microbial composition analysis pipeline for single-molecule real-time 16S rRNA gene sequencing (MCSMRT, https://github.com/jpearl01/mcsmrt) overcomes deficits of standard marker gene based microbiome analyses by using CCS of entire 16S rRNA genes to provide increased taxonomic and phylogenetic resolution. Extensions of this approach to other marker genes could help refine taxonomic assignments of microbial species and improve reference databases, as well as strengthen the specificity of associations between microbial communities and dysbiotic states.

bioinformatics↗

bioRxiv2018

Tissue dependent expression of bitter receptor TAS2R38 mRNA

TAS2R38 is a human bitter receptor gene with a common but inactive allele and people homozygous for the inactive form cannot perceive certain bitter compounds. The frequency of the inactive and active form of this receptor is nearly equal in many human populations and heterozygotes (with one copy of the active form and another copy of the inactive form) have the most common diplotype. However, heterozygotes differ markedly in their perception of bitterness, in part perhaps because of differences in TAS2R38 mRNA expression. Other tissues including the nasal sinuses express this receptor, where it contributes to pathogen defense. We asked whether heterozygotes with high TAS2R38 mRNA expression in taste tissue were also likely to express more TAS2R38 mRNA in sinonasal tissue. To that end, we measured TAS2R38 bitter taste receptor mRNA by qPCR in biopsied tissue, and learned that expression amount of one is not related to the other. We confirmed the general independence of expression in other tissue expressing TAS2R38 mRNA using autopsy data from the GTEx project. Taste tissue TAS2R38 mRNA expression among heterozygotes is unlikely to predict expression in other tissues, perhaps reflecting tissue-dependent function and hence regulation of this protein.

molecular biology↗