We then tested how CquiOR21 would respond to other commercially available repellents, i.e., PMD, IR3535, and picaridin. In these new preparations, CquiOR21/CquiOrcoexpressing oocytes responded to DEET and IR3535 with dose-dependent outward currents . Picaridin elicited minor outward currents at lower doses but robust outward currents at 1 mM dose. By contrast, PMD did not elicit outward currents; it was silent at lower doses and gave minor inward currents at the highest dose, 1 mM . We then interrogated CquiOR21 orthologs from the yellow fever mosquito, AaegOR10 , and the malaria mosquito, AgamOR10 . AaegOR10/AaegOrco- and AgamOR10/AgamOrco-expressing oocytes responded with a similar pattern to that observed with CquiOR21/CquiOrco-expressing oocytes . Specifically, DEET generated dose-dependent outward currents as did picaridin at 1 mM, whereas PMD elicited only minor currents. Over the years, stackable planters we have deorphanized multiple ORs from Cx. quinquefasciatus and were surprised to observe that these outward currents generated only with preparations involving ORs sensitive to oviposition attractants. We then tested other ORs for oviposition attractants, namely CquiOR121 , CquiOR37, and CquiOR99 .
Oocytes expressing each of these ORs along with the obligatory coreceptor Orco elicited dose dependent outward currents when challenged with DEET . We also challenged other ORs from the malaria mosquito, which are not involved in the reception of oviposition attractants. Like their Culex counterparts, ORs unrelated to oviposition attractants did not generate outward currents when challenged with DEET . A previously reported larval OR, AgamOR40 generated dose-dependent inward currents in response to DEET as well as to its best ligand, fenchone . By contrast, oocytes expressing AgamOR8 along with AgamOrco generated robust, dose-dependent inward currents in response to 1-octen-3-ol, but it was silent to DEET . Previously, it has been demonstrated that DEET modulates responses of other odorants to ORs , but no outward currents were recorded. When odorants were present in combination with DEET at high I doses, the odorant-induced inward currents decreased significantly , but DEET per se did not elicit measurable currents. At the time of this writing, a small DEETinduced hyperpolarization of a mosquito OR was reported . Our fortuitous discovery of outward current elicited by DEET might occur mainly on ORs sensitive to oviposition attractants.
However, we have recently reported outward current selicited by multiple compounds, including repellents, on a Culex OR, CquiOR32, which is sensitive to a plant-derived compound with repellency activity, methyl salicylate . It is, therefore, conceivable that the phenomenon expands beyond OR sensitive to mosquito oviposition attractants. Next, we challenged CquiOR21, AaegOR10, and AgamOR10 with a panel of 20 compounds, which includes plant-derived and plant-inspired repellents. The compounds are part of pending worldwide and US patent applications and have been previously tested as oviposition deterrents for an agricultural pest, the navel orangeworm, Amyelois transitella . The panel was provided to the experimenter with code names, i.e., BDR1-20. To make certain the compounds would be properly identified post hoc, one of us analyzed each sample by GC-MS before electrophysiology and behavior work. None of the 20 compounds elicited inward currents , and 4 compounds did not generate measurable currents, specifically BDR-7, 11, 15, and 20, which were later decoded by W.S.L. to the experimenter. They are ethyl palmitate, 2-pentadecanol, dihydrojasmonic acid, and 2-pentadecanone, respectively. Other compounds generated robust outward currents at least in one of the three ORs tested. They are BDR-3 , BDR-4 , BDR-10 , BDR-14 , and BDR-19 .
Of note, repellency activity for methyl jasmonate and methyl dihydrojasmonate has been previously demonstrated. Using AgamOR10/AgamOrco-expressing oocytes , we recorded dose-dependent outward currents generated by these compounds at 0.01, 0.1, and 1 mM. We then investigated whether these outward currents would modulate CquiOR21 responses to skatole. Thus, CquiOR21/CquiOrco-expressing oocytes were challenged with skatole alone or in mixtures with one of the test compounds. Based on preliminary experiments showing that DEET modulates the response to skatole, we selected DEET as a positive control and tested two compounds from our panel, which generated strong/moderate and weak outward currents, i.e., BDR-4 and 5, respectively . Skatole was presented at a constant dose of 0.1 μM, and the tested compounds were added at decreasing doses from 1 mM to 15 μM . When mixtures of skatole and DEET or BDR-4 at high doses were applied, outward currents were recorded, whereas attenuated inward currents were observed with mixtures containing BDR-5 at the same doses . The effect of DEET and BDR-4 on CquiOR21 responses to skatole was clearly dose-dependent. When the test compounds were coapplied at 125 μM or lower, only inward currents were recorded. In the case of DEET and BDR-4, the inward currents were attenuated even when these compounds were presented at the lowest dose of 15 μM . Although this dataset clearly shows that responses to skatole were modulated by DEET , it does not explain the mode of action of DEET as a noncontact disengagent . Mosquitoes responding to CquiOR21 are not host-seeking mosquitoes, but rather gravid females searching for oviposition sites. The observed modulation may explain at least in part the “off-label” activity of DEET as a deterrent for oviposition .Next, we asked whether compounds modulating OR response to oviposition attractants would activate a DEET receptor mediating spatial repellency .Previously, we have identified CquiOR136 as a DEET receptor in the Southern house mosquito , which is activated by the four major commercially available repellents, DEET, PMD, IR3535, and picaridin . CquiOR136/CquiOrcoexpressing oocytes were challenged with our panel at three doses . IR3535, which elicits the strongest responses at 1 mM , was used as a positive control. BDR-3 and BDR-14 , among other compounds, elicited robust inward currents . We then constructed concentration-response relationships for all compounds in our panel . These analyses clearly show that BDR-14 is the best ligand for CquiOR136 from all tested compounds thus far. More importantly, our data show that CquiOR136 is very sensitive to plant-derived compounds . Specifically, CquiOR136/CquiOrcoexpressing oocytes gave robust responses to methyl dihydrojasmolate, methyl dihydrojasmonate, ethyl dihydrojasmonate, dihydrojasminlactone, dihydrojasmindiol, and methyl jasmonate, which are plant metabolites or their derivatives . Methyl dihydrojasmolate is a reduced form of methyl dihydrojasmonate , which in turn is the product of hydrogenation of the plant hormone methyl jasmonate. That this DEET receptor is very sensitive to these plant-derived and plant-inspired compounds is consistent with the notion that the primary function of CquiOR136 in the biology of Cx. quinquefasciatus is the reception of plant defense compounds and that DEET mimics these natural products .Our data suggest that mosquito response to oviposition attractants may be modulated by repellents. When ORs sensitive to oviposition attractants were challenged with repellent, stacking pots outward currents were generated. Responses of the OR detecting the oviposition skatole in the Southern house mosquito, CquiOR21 , were reduced when skatole was coapplied with DEET or methyl dihydrojasmolate. These inhibitory currents may explain at least in part, the deterrent effect of DEET on the attraction of gravid females . Therefore, DEET-mediated oviposition deterrence may have two modes of action . More importantly, the discovery of inhibitory currents demonstrates that the integration of chemical signals at the peripheral olfactory system is more complex than previously appreciated.Blueberries have been widely studied for their high phytonutrient content, particularly phenolic compounds.
Dietary polyphenols found in blueberries consist of flavonoids and phenolic acids . Anthocyanins are the pigments responsible for the color of berries and blueberries have one of the highest anthocyanin contents among foods . The individual anthocyanin profile of blueberries is complex and contains 5 of the 6 anthocyanidins commonly present in food: malvidin, cyanidin, delphinidin, petunidin, and peonidin . The glycoside moieties attached to the anthocyanidin are predominantly galactose, arabinose, and glucose , with all combinations of the 5 anthocyanidins and 3 sugars found across blueberry cultivars. Blueberries are also rich in flavonols, with a predominance of quercetin derivatives , and proanthocyanidins, formed by polymerization of catechin and/or epicatechin units . Nonflavonoid phenolic acids are mainly represented by chlorogenic acid, which results from the esterification of caffeic acid with a quinic acid molecule . Blueberry phytochemicals used in the treatment of cells in vitro often consist of a whole extract, delivered as a reconstituted powder, juice, or pomace , concentrated or not. Specific classes of phytochemicals such as polyphenol-rich extracts and phenolic fractions, including anthocyanins, phenolic acids, and proanthocyanidins are prepared using solvent extraction and purified through solid phase extraction.Polyphenols found in blueberries have been shown to contribute to their health benefits . A number of reviews discuss the association between blueberry consumption and cardiovascular health , inflammatory markers , type 2 diabetes, neuroprotection, and ocular health . These claims on the health benefits of blueberry consumption are supported by epidemiological studies , animal studies , and diverse cell culture models . Randomized controlled trials have investigated antioxidant and anti-inflammatory effects of blueberry in the context of hypertension, cardiovascular diseases, arthritis, insulin resistance, and metabolic syndrome and supplemented with doses between 20 and 50 g of wild blueberry powder, equivalent to 1 to 2 cups of fresh blueberries daily for 6 to 16 wk . However, few reported direct modulation of molecular markers via blueberry supplementation, including circulating inflammatory cytokines and adhesion molecules. To complete and extend the body of literature covering the in vivo physiological effects of blueberry feeding, the current review considers mechanisms of action by focusingon in vitro responses to blueberry components. Evaluation of the bio-active potential of berry phytochemicals or extracts often uses cell models , which serve as controlled, simplified systems . Numerous limitations exist regarding cell culture conditions and the artificial environment in which the cells are maintained, because they are not completely representative of the body’s physiology . In general, cells are treated with parent compounds, disregarding potential host and microbial metabolism between consumption and the moment compounds reach the target organ . Cells are also not always treated with amounts representative of physiological concentrations in the body, which can be low due to the limited absorption of polyphenols . They are relatively simple to access and maintain, and provide insights into the cellular mechanisms of the studied compounds . Although blueberry phytochemicals may impact a multitude of health-related mechanisms, we focused on 2 intrinsically related systems , the regulation of which are central to health, and when dysregulated, underlie many disease outcomes. The objective of this narrative review is to discuss observations related to the modulatory role of blueberry phytochemicals on key pathways implicated in systems, and to consider the results from a physiological perspective.Search queries containing the keywords “inflammation”, “anti-inflammatory”, “oxidative stress”, “cell culture”, “in vitro”, and “berry” were conducted in the PubMed and Science Direct databases. The search was conducted for articles through to August 2021. Duplicates, reviews, articles written in languages other than English, and studies using animal models or human participants were excluded with the caveat that studies focusing on animals and/or humans but containing complementary cell-based experiments were included and an evaluation of the cell model findings used in the current review. From all studies on berries and cell culture-based models retrieved, only studies using blueberries were included. In this article, the term “blueberry” encompasses fruits from the Vaccinium genus described as blueberries, including V. angustifolium, V. corymbosum, V. ashei, V. uliginosum, and the European blueberry also referred to as bilberry,V. myrtillus. In addition, relevant references from earlier reviews were manually entered. A total of 70 articles related to blueberry and cell culture models of inflammation, oxidative stress, and related conditions were included.Inflammation is the innate immune system reaction to a stimulus generated by pathogens, damaged cells, carcinogens, toxic compounds, changes in concentrations of reactive oxygen species , and some foods or metabolites . General response mechanisms of inflammation have been extensively reviewed elsewhere . In brief, the NF-κB and mitogen-activated protein kinase [MAPK, subdivided into extracellular-signal-regulated kinase , c-Jun Nterminal kinase , and p38] inflammatory pathways are activated following an external stimulus and/or by proinflammatory cytokines . Their activation generates the production of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, which upon release mobilize immune cells . Abnormal activation of inflammation-associated proteins, including NADPH oxidase , inducible NO synthase , and cyclooxygenase -2 , and failure to resolve the infection or injury can lead to chronic inflammation linked to diseases and cardiometabolic dysfunction.