Identification of attractants for adult Spodoptera litura based on the interaction between odorant-binding protein 34 and host volatiles.

Odorant-binding proteins (OBPs) play key roles in host plant location by insects, and can accordingly serve as important targets for the development of attractants. In this study, we detected the high expression of SlitOBP34 in male antennae of Spodoptera litura. Subsequently, the fluorescence competitive binding experiments displayed that the SlitOBP34 protein has binding affinity for different ligands. Then, protein-ligand interaction analyses found the presence of six amino acid residues may serve as key recognition sites. Further electroantennographic and biobehavioral assessments revealed that the electrophysiological responses of male antennae were evoked in response to stimulation with the six identified host volatiles, and that these volatiles attracted male moths to varying extents. Notably, low concentrations of benzaldehyde, 1-hexanol, and cis-3-hexenyl acetate were found to have significant attractant effects on male moths, thereby identifying these three host volatiles as potential candidates for the development of male attractants. These findings advance our current understanding of the olfactory-encoded mechanisms of host plants selection in S. litura and have enabled us to develop novel adult attractants for controlling the pest in the future.

Safety, Pharmacokinetics, and Pharmacodynamics of the Oral TLR8 Agonist Selgantolimod in Chronic Hepatitis B.

BACKGROUND AND AIMS: In patients with chronic hepatitis B (CHB) infection, activation of toll-like receptor 8 may induce antiviral immunity and drive functional cure. Selgantolimod, a toll-like receptor 8 agonist, was evaluated in patients with CHB who were virally suppressed on oral antiviral treatment or viremic and not on oral antiviral treatment. APPROACH AND RESULTS: In this phase 1b study, patients were randomized 4:1 to receive either selgantolimod or placebo once weekly. Virally suppressed patients received either 1.5 mg (for 2 weeks) or 3 mg (for 2 weeks or 4 weeks). Viremic patients received 3 mg for 2 weeks. The primary endpoint was safety, as assessed by adverse events (AEs), laboratory abnormalities, and vital sign examination. Pharmacokinetic and pharmacodynamic parameters were assessed by plasma analysis. A total of 38 patients (28 virally suppressed, 10 viremic) were enrolled from six sites in Australia, New Zealand, and South Korea. Twenty patients (53%) experienced an AE and 32 (84%) had laboratory abnormalities, all of which were mild or moderate in severity. The most common AEs were headache (32%), nausea (24%), and dizziness (13%). With a half-life of 5 hours, no accumulation of selgantolimod was observed with multiple dosing. Selgantolimod induced transient dose-dependent increases in serum cytokines, including IL-12p40 and IL-1RA, which are important for the expansion and activity of multiple T- cell subsets and innate immunity. CONCLUSION: Selgantolimod was safe and well-tolerated in virally suppressed and viremic patients with CHB and elicited cytokine responses consistent with target engagement. Further studies with longer durations of selgantolimod treatment are required to evaluate efficacy.

Toll-Like Receptor 8 Agonist GS-9688 Induces Sustained Efficacy in the Woodchuck Model of Chronic Hepatitis B.

BACKGROUND AND AIMS: GS-9688 (selgantolimod) is an oral selective small molecule agonist of toll-like receptor 8 in clinical development for the treatment of chronic hepatitis B. In this study, we evaluated the antiviral efficacy of GS-9688 in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to hepatitis B virus. APPROACH AND RESULTS: WHV-infected woodchucks received eight weekly oral doses of vehicle, 1 mg/kg GS-9688, or 3 mg/kg GS-9688. Vehicle and 1 mg/kg GS-9688 had no antiviral effect, whereas 3 mg/kg GS-9688 induced a >5 log10 reduction in serum viral load and reduced WHV surface antigen (WHsAg) levels to below the limit of detection in half of the treated woodchucks. In these animals, the antiviral response was maintained until the end of the study (>5 months after the end of treatment). GS-9688 treatment reduced intrahepatic WHV RNA and DNA levels by >95% in animals in which the antiviral response was sustained after treatment cessation, and these woodchucks also developed detectable anti-WHsAg antibodies. The antiviral efficacy of weekly oral dosing with 3 mg/kg GS-9688 was confirmed in a second woodchuck study. The antiviral response to GS-9688 did not correlate with systemic GS-9688 or cytokine levels but was associated with transient elevation of liver injury biomarkers and enhanced proliferative response of peripheral blood mononuclear cells to WHV peptides. Transcriptomic analysis of liver biopsies taken prior to treatment suggested that T follicular helper cells and various other immune cell subsets may play a role in the antiviral response to GS-9688. CONCLUSIONS: Finite, short-duration treatment with a clinically relevant dose of GS-9688 is well tolerated and can induce a sustained antiviral response in WHV-infected woodchucks; the identification of a baseline intrahepatic transcriptional signature associated with response to GS-9688 treatment provides insights into the immune mechanisms that mediate this antiviral effect.

Therapeutic Potential of TLR8 Agonist GS-9688 (Selgantolimod) in Chronic Hepatitis B: Remodeling of Antiviral and Regulatory Mediators.

BACKGROUND AND AIMS: GS-9688 (selgantolimod) is a toll-like receptor 8 agonist in clinical development for the treatment of chronic hepatitis B (CHB). Antiviral activity of GS-9688 has previously been evaluated in vitro in HBV-infected hepatocytes and in vivo in the woodchuck model of CHB. Here we evaluated the potential of GS-9688 to boost responses contributing to viral control and to modulate regulatory mediators. APPROACH AND RESULTS: We characterized the effect of GS-9688 on immune cell subsets in vitro in peripheral blood mononuclear cells of healthy controls and patients with CHB. GS-9688 activated dendritic cells and mononuclear phagocytes to produce IL-12 and other immunomodulatory mediators, inducing a comparable cytokine profile in healthy controls and patients with CHB. GS-9688 increased the frequency of activated natural killer (NK) cells, mucosal-associated invariant T cells, CD4+ follicular helper T cells, and, in about 50% of patients, HBV-specific CD8+ T cells expressing interferon-γ. Moreover, in vitro stimulation with GS-9688 induced NK-cell expression of interferon-γ and TNF-α, and promoted hepatocyte lysis. We also assessed whether GS-9688 inhibited immunosuppressive cell subsets that might enhance antiviral efficacy. Stimulation with GS-9688 reduced the frequency of CD4+ regulatory T cells and monocytic myeloid-derived suppressor cells (MDSCs). Residual MDSCs expressed higher levels of negative immune regulators, galectin-9 and programmed death-ligand 1. Conversely, GS-9688 induced an expansion of immunoregulatory TNF-related apoptosis-inducing ligand+ NK cells and degranulation of arginase-I+ polymorphonuclear MDSCs. CONCLUSIONS: GS-9688 induces cytokines in human peripheral blood mononuclear cells that are able to activate antiviral effector function by multiple immune mediators (HBV-specific CD8+ T cells, CD4+ follicular helper T cells, NK cells, and mucosal-associated invariant T cells). Although reducing the frequency of some immunoregulatory subsets, it enhances the immunosuppressive potential of others, highlighting potential biomarkers and immunotherapeutic targets to optimize the antiviral efficacy of GS-9688.

Trimethylamine N-oxide promotes hyperlipidemia acute pancreatitis via inflammatory response.

Trimethylamine N-oxide (TMAO), a metabolite of gut microbiota, is involved in the regulation of lipid metabolism and inflammatory response; however, the role of TMAO in hyperlipidemia acute pancreatitis (HAP) is not clear. In this study, HAP mice were used as an animal model to explore the effects and possible mechanism of TMAO on HAP, which may provide new ideas for the treatment of HAP. Results found that the levels of triglycerides, total cholesterol, low-density lipoprotein cholesterol, nonestesterified fatty acid, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, α-amylase, TMAO, and flavin-containing monooxygenase 3 were significantly increased, the levels of high-density lipoprotein cholesterol and insulin were significantly decreased, and there was an obvious pancreatic injury and inflammatory response in the model group. The choline analogue 3,3-dimethyl-1-butanol (DMB) treatment reversed the changes of serum biochemical parameters, alleviated the pancreatic tissue injury, and reduced the levels of inflammatory cytokines. Further studies of toll-like receptor (TLR)/p-glycoprotein 65 (p65) pathway found that the expressions of TLR2, TLR4, and p-p65/p65 in the model group were significantly increased, which was more obvious after Escherichia coli (Migula) Castellani & Chalmers treatment, while activation of the TLR/p65 pathway was inhibited by DMB. The results indicated that TMAO promotes HAP by promoting inflammatory response through TLR/p65 signaling pathway, suggesting that TMAO may be a potential target of HAP.

Sex Pheromone of the Alfalfa Plant Bug, Adelphocoris lineolatus: Pheromone Composition and Antagonistic Effect of 1-Hexanol (Hemiptera: Miridae).

The sex pheromone composition of alfalfa plant bugs, Adelphocoris lineolatus (Goeze), from Central Europe was investigated to test the hypothesis that insect species across a wide geographical area can vary in pheromone composition. Potential interactions between the pheromone and a known attractant, (E)-cinnamaldehyde, were also assessed. Coupled gas chromatography-electroantennography (GC-EAG) using male antennae and volatile extracts collected from females, previously shown to attract males in field experiments, revealed the presence of three physiologically active compounds. These were identified by coupled GC/mass spectrometry (GC/MS) and peak enhancement as hexyl butyrate, (E)-2-hexenyl butyrate and (E)-4-oxo-2-hexenal. A ternary blend of these compounds in a 5.4:9.0:1.0 ratio attracted male A. lineolatus in field trials in Hungary. Omission of either (E)-2-hexenyl-butyrate or (E)-4-oxo-2-hexenal from the ternary blend or substitution of (E)-4-oxo-2-hexenal by (E)-2-hexenal resulted in loss of activity. These results indicate that this Central European population is similar in pheromone composition to that previously reported for an East Asian population. Interestingly, another EAG-active compound, 1-hexanol, was also present in female extract. When 1-hexanol was tested in combination with the ternary pheromone blend, male catches were reduced. This compound showed a dose-response effect with small doses showing a strong behavioral effect, suggesting that 1-hexanol may act as a sex pheromone antagonist in A. lineolatus. Furthermore, when (E)-cinnamaldehyde was field tested in combination with the sex pheromone, there was no increase in male catch, but the combination attracted both males and females. Prospects for practical application are discussed.

Butanol is cytotoxic to Lactococcus lactis while ethanol and hexanol are cytostatic.

Lactic acid bacteria currently used extensively by the dairy industry have a superior tolerance towards short-chain alcohols, which makes them interesting targets for use in future bio-refineries. The mechanism underlying the alcohol tolerance of lactic acid bacteria has so far received little attention. In the present study, the physiological alcohol stress response of Lactococcus lactis subsp. cremoris MG1363 towards the primary, even-chain alcohols ethanol, butanol and hexanol, was characterized. The alcohol tolerance of L. lactis was found to be comparable to those reported for highly alcohol-resistant lactic acid bacteria. Combined results from alcohol survival rate, live/dead staining, and a novel usage of the ß-galactosidase assay, revealed that while high concentrations of ethanol and hexanol were cytostatic to L. lactis, high concentrations of butanol were cytotoxic, causing irreparable damages to the cell membrane.

Responses of Human Neonates to Highly Diluted Odorants from Sweat.

Conjugated forms of odorants contributing to sweat odor occur not only in human sweat but also in amniotic fluid, colostrum, and milk. However, it is unclear whether the released odorants are detected and hedonically discriminated by human newborns. To investigate this issue, we administered highly diluted solutions of (R)/(S)-3-methyl-3-sulfanylhexan-1-ol (MSH), (R)/(S)-3-sulfanylhexan-1-ol (SH), (E)/(Z)-3-methylhex-2-enoic acid (3M2H), and (R)/(S)-3-hydroxy-3-methylhexanoic acid (HMHA) to 3-d-old infants while their respiratory rate and oro-facial movements were recorded. Adult sensitivity to these odorants was assessed via triangle tests. Whereas no neonatal stimulus-specific response was found for respiratory rate, oro-facial reactivity indicated orthonasal detection of MSH and SH by male neonates, and of HMHA by the whole group of neonates. Dependent on the dilution of odorants, newborns evinced neutral responses or longer negative oro-facial expressions compared with the reference stimuli. Finally, newborns appeared to be more sensitive to the target odorants than did adults.

Naturally Produced Defensive Alkenal Compounds Activate TRPA1.

(E)-2-alkenals are aldehydes containing an unsaturated bond between the alpha and beta carbons. 2-alkenals are produced by many organisms for defense against predators and secretions containing (E)-2-alkenals cause predators to stop attacking and allow the prey to escape. Chemical ecologists have described many alkenal compounds with 3-20 carbons common, having varied positions of double bonds and substitutions. How do these defensive alkenals act to deter predators? We have tested the effects of (E)-2-alkenals with 6-12 carbons on transient receptor potential channels (TRP) commonly found in sensory neurons. We find that (E)-2-alkenals activate transient receptor potential ankyrin subtype 1 (TRPA1) at low concentrations-EC50s 10-100 µM (in 0 added Ca(2+) external solutions). Other TRP channels were either weakly activated (TRPV1, TRPV3) or insensitive (TRPV2, TRPV4, TRPM8). (E)-2-alkenals may activate TRPA1 by modifying cysteine side chains. However, target cysteines include others beyond the 3 in the amino-terminus implicated in activation, as a channel with cysteines at 621, 641, 665 mutated to serine responded robustly. Related chemicals, including the aldehydes hexanal and decanal, and (E)-2-hexen-1-ol also activated TRPA1, but with weaker potency. Rat trigeminal nerve recordings and behavioral experiments showed (E)-2-hexenal was aversive. Our results suggest that TRPA1 is likely a major target of these commonly used defensive chemicals.

The residue I257 at S4-S5 linker in KCNQ1 determines KCNQ1/KCNE1 channel sensitivity to 1-alkanols.

AIM: KCNQ1 and KCNE1 form a complex in human ventricular cardiomyocytes, which are important in maintaining a normal heart rhythm. In the present study we investigated the effects of a homologous series of 1-alkanols on KCNQ1/KCNE1 channels expressed in Xenopus oocytes. METHODS: ECG recording was made in rats injected with ethanol-containing solution (0.3 mL, ip). Human KCNQ1 channel and its auxiliary subunit KCNE1 were heterologously coexpressed in Xenopus oocytes, which were superfused with ND96 solution; 1-alkanols (ethanol, 1-butanol and 1-hexanol) were delivered through a gravity-driven perfusion device. The slow-delayed rectifier potassium currents IKs (KCNQ1/KCNE1 currents) were recorded using a two-electrode voltage clamp method. Site-directed mutations (I257A) were made in KCNQ1. RESULTS: In ECG recordings, a low concentration of ethanol (3%, v/v) slightly increased the heart rate of rats, whereas the higher concentrations of ethanol (10%, 50%, v/v) markedly reduced it. In oocytes coexpressing KCNQ1/KCNE1 channels, ethanol, 1-butanol and 1-hexanol dose-dependently inhibited IKs currents with IC50 values of 80, 11 and 2.7 mmol/L, respectively. Furthermore, the 1-alkanols blocked the KCNQ1 channel in both open and closed states, and a four-state model could adequately explain the effects of 1-alkanols on the closed-state channel block. Moreover, the mutation of I257A at the intracellular loop between S4 and S5 in KCNQ1 greatly decreased the sensitivity to 1-alkanols; and the IC50 values of ethanol, 1-butanol and 1-hexanol were increased to 634, 414 and 7.4 mmol/L, respectively. The mutation also caused the ablation of closed-state channel block. CONCLUSION: These findings provide new insight into the intricate mechanisms of the blocking effects of ethanol on the KCNQ1 channel.

Modification of CO2 avoidance behaviour in Drosophila by inhibitory odorants.

The fruitfly Drosophila melanogaster exhibits a robust and innate olfactory-based avoidance behaviour to CO(2), a component of odour emitted from stressed flies. Specialized neurons in the antenna and a dedicated neuronal circuit in the higher olfactory system mediate CO(2) detection and avoidance. However, fruitflies need to overcome this avoidance response in some environments that contain CO(2) such as ripening fruits and fermenting yeast, which are essential food sources. Very little is known about the molecular and neuronal basis of this unique, context-dependent modification of innate olfactory avoidance behaviour. Here we identify a new class of odorants present in food that directly inhibit CO(2)-sensitive neurons in the antenna. Using an in vivo expression system we establish that the odorants act on the Gr21a/Gr63a CO(2) receptor. The presence of these odorants significantly and specifically reduces CO(2)-mediated avoidance behaviour, as well as avoidance mediated by 'Drosophila stress odour'. We propose a model in which behavioural avoidance to CO(2) is directly influenced by inhibitory interactions of the novel odours with CO(2) receptors. Furthermore, we observe differences in the temporal dynamics of inhibition: the effect of one of these odorants lasts several minutes beyond the initial exposure. Notably, animals that have been briefly pre-exposed to this odorant do not respond to the CO(2) avoidance cue even after the odorant is no longer present. We also show that related odorants are effective inhibitors of the CO(2) response in Culex mosquitoes that transmit West Nile fever and filariasis. Our findings have broader implications in highlighting the important role of inhibitory odorants in olfactory coding, and in their potential to disrupt CO(2)-mediated host-seeking behaviour in disease-carrying insects like mosquitoes.

Inhibition of Pseudogymnoascus destructans growth from conidia and mycelial extension by bacterially produced volatile organic compounds.

The recently identified causative agent of white-nose syndrome (WNS), Pseudogymnoascus destructans, has been implicated in the mortality of an estimated 5.5 million North American bats since its initial documentation in 2006 (Frick et al. in Science 329:679-682, 2010). In an effort to identify potential biological and chemical control options for WNS, 6 previously described bacterially produced volatile organic compounds (VOCs) were screened for anti-P. destructans activity. The compounds include decanal; 2-ethyl-1-hexanol; nonanal; benzothiazole; benzaldehyde; andN,N-dimethyloctylamine. P. destructans conidia and mycelial plugs were exposed to the VOCs in a closed air space at 15 and 4 °C and then evaluated for growth inhibition. All VOCs inhibited growth from conidia as well as inhibiting radial mycelial extension, with the greatest effect at 4 °C. Studies of the ecology of fungistatic soils and the natural abundance of the fungistatic VOCs present in these environments suggest a synergistic activity of select VOCs may occur. The evaluation of formulations of two or three VOCs at equivalent concentrations was supportive of synergistic activity in several cases. The identification of bacterially produced VOCs with anti-P. destructans activity indicates disease-suppressive and fungistatic soils as a potentially significant reservoir of biological and chemical control options for WNS and provides wildlife management personnel with tools to combat this devastating disease.

Antifungal Activity and Mechanisms of 2-Ethylhexanol, a Volatile Organic Compound Produced by Stenotrophomonas sp. NAU1697, against Fusarium oxysporum f. sp. cucumerinum.

Researchers often consider microorganisms from Stenotrophomonas sp. to be beneficial for plants. In this study, the biocidal effects and action mechanisms of volatile organic compounds (VOCs) produced by Stenotrophomonas sp. NAU1697 were investigated. The mycelial growth and spore germination of Fusarium oxysporum f. sp. cucumerinum (FOC), which is a pathogen responsible for cucumber wilt disease, were significantly inhibited by VOCs emitted from NAU1697. Among the VOCs, 33 were identified, 11 of which were investigated for their antifungal properties. Among the tested compounds, 2-ethylhexanol exhibited the highest antifungal activity toward FOC, with a minimum inhibitory volume (MIV) of 3.0 µL/plate (equal to 35.7 mg/L). Damage to the hyphal cell wall and cell membrane integrity caused a decrease in the ergosterol content and a burst of reactive oxygen species (ROS) after 2-ethylhexanol treatment. DNA damage, which is indicative of apoptosis-like cell death, was monitored in 2-ethylhexanol-treated FOC cells by using micro-FTIR analysis. Furthermore, the activities of mitochondrial dehydrogenases and mitochondrial respiratory chain complex III in 2-ethylhexanol-treated FOC cells were significantly decreased. The transcription levels of genes associated with redox reactions and the cell wall integrity (CWI) pathway were significantly upregulated, thus indicating that stress was caused by 2-ethylhexanol. The findings of this research provide a new avenue for the sustainable management of soil-borne plant fungal diseases.

Environmental and seasonal influences on red raspberry flavour volatiles and identification of quantitative trait loci (QTL) and candidate genes.

Raspberry volatiles are important for perceptions of sensory quality, mould resistance and some have nutraceutical activities. Twelve raspberry character volatiles were quantified, 11 of them in fruit from two seasons, from plants from the Glen Moy × Latham mapping population growing in both open field and under cover (polytunnels). Effects of season and environment were examined for their impact on the content of α-ionone, α-ionol, ß-ionone, ß-damascenone, linalool, geraniol, benzyl alcohol, (Z)-3-hexenol, acetoin, acetic and hexanoic acids, whilst raspberry ketone was measured in one season. A significant variation was observed in fruit volatiles in all progeny between seasons and method of cultivation. Quantitative trait loci were determined and mapped to six of the seven linkage groups, as were candidate genes in the volatiles pathways.

Structure-activity relationship of volatile compounds that induce defense-related genes in maize seedlings.

Volatile organic compounds mediate plant-to-plant communication, and plants receiving volatile cues can acquire greater defenses against attackers. It has been expected that volatiles are received by factors that eventually lead to the induction of defense-related gene expression; however, the nature of these factors remain unclear. Structure-activity relationship analysis of gene expression induction by volatiles should provide insights into the nature of these factors. We conducted a structure-activity relationship study using maize seedlings and (Z)-3-hexen-1-yl acetate (Z3HAC) as the lead compound. The acid portion of Z3HAC was not essential, and (Z)-3-hexen-1-ol (Z3HOL), which is formed after the hydrolysis of Z3HAC, is likely the structure essential for the upregulation of the genes. The double bond of Z3HOL is essential; however, its geometry is indistinguishable. Strict specificity was detected regarding the length of the methylene chain on the α- and ω-sides of the double bond, and therefore, the 3-hexen-1-ol structure was found to be the ultimate structure. This finding provides insight into the nature of the factors that interact with a volatile compound and subsequently activate signaling pathways, leading to the upregulation of a subset of defense genes.

6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio) Hexanol Inhibits Proliferation and Induces Apoptosis of Endometriosis by Regulating Glutathione S-Transferase Mu Class 4.

Endometriosis is a chronic disease associated with a disrupted oxidative balance and chronic inflammation. In this study, we investigated the role of glutathione S-transferase Mu class 4 (GSTM4) in endometriosis and determined whether 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX) regulates GSTM4 expression to affect cellular functions and oxidative stress. GSTM4 expression was detected by immunohistochemistry in endometrium from 15 endometriosis patients and 15 healthy controls. Western blotting was used to detect the expression of GSTM4, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase-9 (MMP-9), Survivin, B-cell lymphoma-extra-large (Bcl-XL), Bax, kelch-like ECH-associated protein 1 (Keap1), and nuclear factor-erythroid 2-related factor 2 (Nrf2) in primary endometrial stromal cells with endometriosis (EESC) and normal endometrial stromal cells (NESC). The effects of NBDHEX on cell proliferation, migration, and invasion were evaluated using Cell Counting Kit-8 (CCK8) and Transwell assays. Apoptosis was detected by flow cytometry. The expression of GSTM4 was significantly increased in endometrium from endometriosis patients. Upon NBDHEX treatment, ESC exhibited reduced proliferation, migration and invasion abilities, and increased apoptosis. NBDHEX decreased the expression of endometriosis prognostic markers (PCNA and MMP-9) and anti-apoptotic proteins (Survivin and Bcl-xl), while it increased the expression of the apoptotic protein Bax. It had no effect on Keap1 expression, and it decreased the expression of Nrf2. The effect of siRNA-mediated knockdown of GSTM4 was similar to that of suppressing GSTM4 expression with NBDHEX treatment. These results indicate that GSTM4 is highly expressed in endometriosis and its expression is inhibited by NBDHEX. Decreased expression of GSTM4 inhibits cell growth, migration, and invasion, and negatively regulates Nrf2 to affect oxidative stress-induced apoptosis. Our results suggest that GSTM4 may play a role in ameliorating the progression of endometriosis. NBDHEX may have therapeutic potential in the treatment of endometriosis.

Primary alcohols activate human TRPA1 channel in a carbon chain length-dependent manner.

Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable non-selective cation channel that is mainly expressed in primary nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent compounds such as mustard oil and cinnamaldehyde, and intracellular alkalization. Here, we show that primary alcohols, which have been reported to cause skin, eye or nasal irritation, activate human TRPA1 (hTRPA1). We measured intracellular Ca(2+) changes in HEK293 cells expressing hTRPA1 induced by 1 mM primary alcohols. Higher alcohols (1-butanol to 1-octanol) showed Ca(2+) increases proportional to the carbon chain length. In whole-cell patch-clamp recordings, higher alcohols (1-hexanol to 1-octanol) activated hTRPA1 and the potency increased with the carbon chain length. Higher alcohols evoked single-channel opening of hTRPA1 in an inside-out configuration. In addition, cysteine at 665 in the N terminus and histidine at 983 in the C terminus were important for hTRPA1 activation by primary alcohols. Furthermore, straight-chain secondary alcohols increased intracellular Ca(2+) concentrations in HEK293 cells expressing hTRPA1, and both primary and secondary alcohols showed hTRPA1 activation activities that correlated highly with their octanol/water partition coefficients. On the other hand, mouse TRPA1 did not show a strong response to 1-hexanol or 1-octanol, nor did these alcohols evoke significant pain in mice. We conclude that primary and secondary alcohols activate hTRPA1 in a carbon chain length-dependent manner. TRPA1 could be a sensor of alcohols inducing skin, eye and nasal irritation in human.

Evolutionary grass roots for odor recognition.

Considerable evidence supports the idea that odorant recognition depends on specific sequence variations in olfactory receptor (OR) proteins. Much of this emerges from in vitro screens in heterogenous expression systems. However, the ultimate proof should arise from measurements of odorant thresholds in human individuals harboring different OR genetic variants, a research vein that has so far been only scantly explored. The study of McRae et al., published in this issue of Chemical Senses, shows how the recognition of a grassy odorant depends on specific OR interindividual sequence changes. It provides a clear relevant example for the impact of genetics on olfaction and is an excellent portrayal of the power of human genomics to decipher olfactory perception.

Genetic variation in the odorant receptor OR2J3 is associated with the ability to detect the "grassy" smelling odor, cis-3-hexen-1-ol.

The ability to detect many odors varies among individuals; however, the contribution of genotype to this variation has been assessed for relatively few compounds. We have identified a genetic basis for the ability to detect the flavor compound cis-3-hexen-1-ol. This compound is typically described as "green grassy" or the smell of "cut grass," with variation in the ability to detect it linked to single nucleotide polymorphisms (SNPs) in a region on human chromosome 6 containing 25 odorant receptor genes. We have sequenced the coding regions of all 25 receptors across an ethnically mixed population of 52 individuals and identified 147 sequence variants. We tested these for association with cis-3-hexen-1-ol detection thresholds and found 3 strongly associated SNPs, including one found in a functional odorant receptor (rs28757581 in OR2J3). In vitro assays of 13 odorant receptors from the region identified 3 receptors that could respond to cis-3-hexen-1-ol, including OR2J3. This gene contained 5 predicted haplotypes across the 52 individuals. We tested all 5 haplotypes in vitro and several amino acid substitutions on their own, such as rs28757581 (T113A). Two amino acid substitutions, T113A and R226Q, impaired the ability of OR2J3 to respond to cis-3-hexen-1-ol, and together these two substitutions effectively abolished the response to the compound. The haplotype of OR2J3 containing both T113A and R226Q explains 26.4% of the variation in cis-3-hexen-1-ol detection in our study cohort. Further research is required to examine whether OR2J3 haplotypes explain variation in perceived flavor experience and the consumption of foods containing cis-3-hexen-1-ol.

Modulation by octopamine of olfactory responses to nonpheromone odorants in the cockroach, Periplaneta americana L.

Olfactory receptor cells in insects are modulated by neurohormones. Recordings from cockroach olfactory sensilla showed that a subset of sensory neurons increase their responses to selected nonpheromone odorants after octopamine application. With octopamine application, recordings demonstrated increased firing rates by the short but not the long alcohol-sensitive sensilla to the nonpheromone volatile, hexan-1-ol. Within the same sensillum, individual receptor cells are shown to be modulated independently from each other, indicating that the octopamine receptors reside in the receptor not in the accessory cells. A uniform decrease in the amplitude of electroantennogram, which is odorant independent, is suggested to reflect the rise in octopamine concentration in the antennal hemolymph. Perception of general odorants measured as behavioral responses changed qualitatively under octopamine treatment: namely, repulsive hexan-1-ol became neutral, whereas neutral eucalyptol became attractive. Octopamine induced a change in male behavioral responses to general odors that were essentially the same as in the state of sexual arousal. Our findings suggest that sensitivity to odors having different biological significances is modulated selectively at the peripheral as well as other levels of olfactory processing.