The structural basis of odorant recognition in insect olfactory receptors.

Olfactory systems must detect and discriminate amongst an enormous variety of odorants1. To contend with this challenge, diverse species have converged on a common strategy in which odorant identity is encoded through the combinatorial activation of large families of olfactory receptors1-3, thus allowing a finite number of receptors to detect a vast chemical world. Here we offer structural and mechanistic insight into how an individual olfactory receptor can flexibly recognize diverse odorants. We show that the olfactory receptor MhOR5 from the jumping bristletail4 Machilis hrabei assembles as a homotetrameric odorant-gated ion channel with broad chemical tuning. Using cryo-electron microscopy, we elucidated the structure of MhOR5 in multiple gating states, alone and in complex with two of its agonists-the odorant eugenol and the insect repellent DEET. Both ligands are recognized through distributed hydrophobic interactions within the same geometrically simple binding pocket located in the transmembrane region of each subunit, suggesting a structural logic for the promiscuous chemical sensitivity of this receptor. Mutation of individual residues lining the binding pocket predictably altered the sensitivity of MhOR5 to eugenol and DEET and broadly reconfigured the receptor's tuning. Together, our data support a model in which diverse odorants share the same structural determinants for binding, shedding light on the molecular recognition mechanisms that ultimately endow the olfactory system with its immense discriminatory capacity.

A natural variant and engineered mutation in a GPCR promote DEET resistance in C. elegans.

DEET (N,N-diethyl-meta-toluamide) is a synthetic chemical identified by the US Department of Agriculture in 1946 in a screen for repellents to protect soldiers from mosquito-borne diseases1,2. Since its discovery, DEET has become the world's most widely used arthropod repellent and is effective against invertebrates separated by millions of years of evolution-including biting flies3, honeybees4, ticks5, and land leeches3. In insects, DEET acts on the olfactory system5-12 and requires the olfactory receptor co-receptor Orco7,9-12, but exactly how it works remains controversial13. Here we show that the nematode Caenorhabditis elegans is sensitive to DEET and use this genetically tractable animal to study the mechanism of action of this chemical. We found that DEET is not a volatile repellent, but instead interferes selectively with chemotaxis to a variety of attractant and repellent molecules. In a forward genetic screen for DEET-resistant worms, we identified a gene that encodes a single G protein-coupled receptor, str-217, which is expressed in a single pair of chemosensory neurons that are responsive to DEET, called ADL neurons. Mis-expression of str-217 in another chemosensory neuron conferred responses to DEET. Engineered str-217 mutants, and a wild isolate of C. elegans that carries a str-217 deletion, are resistant to DEET. We found that DEET can interfere with behaviour by inducing an increase in average pause length during locomotion, and show that this increase in pausing requires both str-217 and ADL neurons. Finally, we demonstrated that ADL neurons are activated by DEET and that optogenetic activation of ADL neurons increased average pause length. This is consistent with the 'confusant' hypothesis, which proposes that DEET is not a simple repellent but that it instead modulates multiple olfactory pathways to scramble behavioural responses10,11. Our results suggest a consistent motif in the effectiveness of DEET across widely divergent taxa: an effect on multiple chemosensory neurons that disrupts the pairing between odorant stimulus and behavioural response.

An updated review on the safety of N, N-diethyl-meta-toluamide insect repellent use in children and the efficacy of natural alternatives.

N, N-diethyl-meta-toluamide (DEET) has been considered the 'gold standard' for insect repellent use since the 1950s and constitutes most insect repellents on the market. However, conflicting data in the scientific literature and confusing information in the media are at the core of debates about the safety of DEET insect repellents for the protection of children against arthropod bites. The few fatal occurrences involving DEET insect repellents and complications of their use in the pediatric population are typically the result of accidental overdoses or misuse of insect repellents that disregard warnings on product labels. With appropriate application, the safety record of DEET insect repellents continues to be excellent with few side effects. The purpose of this review is to provide a summary of the literature on safety outcomes of DEET insect repellent use in children; outline the pediatric recommendations relating to DEET insect repellents; and provide an overview of EPA-approved and naturally derived alternatives to DEET that possess low toxicity while providing a similar level of protection to synthetic insect repellents.

Recent advances in degradation of N,N-diethyl-3-toluamide (DEET)-an emerging environmental contaminant: a review.

N,N-Diethyl-3-toluamide (DEET) is a commonly used insect repellent, which acts as an organic chemical contaminant in water and considered as an emerging contaminant which has been observed worldwide. It gets discharged into the environment through sewage waste. The various methods have been used to degrade DEET, such as UV based, ozonation, photocatalytic degradation, and biodegradation (based on the metabolic activity of fungi and bacteria). However, less research has been done on the degradation of DEET by deploying nanoparticles. Therefore, biodegradation and nanotechnology-based methods can be the potential solution to remediate DEET from the environment. This review is an attempt to analyze the routes of entry of DEET into the atmosphere and its environmental health consequences and to explore physical, chemical, and biological methods of degradation. Furthermore, it focuses on the various methods used for the biodegradation of the DEET, including their environmental consequences. Future research is needed with the application of biological methods for the degradation of DEET. Metabolic pathway for biodegradation was explored for the new potent microbial strains by the application of physical, chemical, and microbial genomics; molecular biology; genetic engineering; and genome sequencing methods.

Amine-Assisted Simultaneous CO2 Absorption and Mineral Carbonation: Effect of Different Categories of Amines.

The hybrid technology of CO2 capture-mineral carbonation (CCMC) using alkaline streams has emerged in recent years. However, thus far, there has been no comprehensive study revealing the mechanisms of the simultaneous CCMC process regarding the choice of amine types and sensitivity of parameters. Combining with the analysis of multistep reaction mechanisms for different amines, we investigated a representative from each category in CCMC using calcium chloride to simulate the alkaline resource after leaching, i.e., primary (ethanolamine, MEA), secondary (diisopropanolamine, DIPA), tertiary (diethylethanolamine, DEAE), and triamine (diethylenetriamine, DETA), respectively. In the adsorption step, increasing the amine concentration beyond 2 mol/L reduced the absorption efficiency of DEAE due to the hydration mechanism, motivating a rational choice of concentration. In CCMC sections, when the amine concentration increased, only DEAE exhibited an increased carbonation efficiency of up to 100%, while DETA showed the lowest conversion. The carbonation of DEAE demonstrated the least sensitivity to temperature. The crystal transformation experiments suggested that over time, the produced vaterite could completely transform to calcite or aragonite, except those from DETA. Thus, with rationally chosen conditions, DEAE was demonstrated ideal for CCMC. These findings obtained in this work provided a theoretical foundation for designing future CCMC processes.

Generation of Reactive Nitrogen Species in UV Photolysis of Dichloramine and Their Incorporation into Nitrogenous Byproducts.

Dichloramine (NHCl2) often coexists with monochloramine (NH2Cl) in reverse osmosis (RO) permeate in potable reuse scenarios when NH2Cl is added upstream of RO for membrane fouling control such that UV photolysis of NHCl2 occurs during the downstream UV/chloramine process. However, the formation of reactive nitrogen species (RNS) and their incorporation into byproducts during the UV/NHCl2 process are largely unknown. This study quantitatively evaluated the generation of RNS in the UV/NHCl2 process and investigated the role of RNS in micropollutant transformation. UV photolysis of NHCl2 produced comparable RNS concentration to that of NH2Cl at the same oxidant dosage (100 µM) at pH 5.5. Under the experimental conditions, the RNS contributed greatly (40.6%) to N,N-diethyl-3-methylbenzamide (DEET) degradation. By using 15N-labeling and mass spectrometry methods, seven nitrogenous byproducts of DEET degradation with the incorporation of nitrogen originating from the RNS were detected. Among these seven byproducts, six were identified to contain a nitro group (-NO2). While the UV/NHCl2 process formed comparable intensities of -NO-containing products to those in the UV/NH2Cl process, the later process formed 3-91% higher intensities of -NO2-containing products. These findings are essential in furthering our understanding of the contribution of the UV/NHCl2 process in potable reuse scenarios.

Insect repellents: An updated review for the clinician.

Malaria, Zika virus, West Nile virus, Dengue fever, and Lyme disease are common causes of morbidity and mortality around the world. While arthropod bites may cause local inflammation and discomfort, a greater concern is the potential to develop deadly systemic infection. The use of insect repellents (IRs) to prevent systemic infections constitutes a fundamental public health effort. Cost effectiveness, availability, and high efficacy against arthropod vectors are key characteristics of an ideal IR. Currently, numerous IRs are available on the market, with N,N-diethyl-3-methylbenzamide (DEET) being the most widely used. DEET has an excellent safety profile and remarkable protection against mosquitoes and various other arthropods. Other Environmental Protection Agency-registered IR ingredients (eg, permethrin, picaridin, IR3535, oil of lemon eucalyptus, oil of citronella, catnip oil, and 2-undecanone) are alternative IRs of great interest because some of these ingredients have efficacies comparable to that of DEET. These alternative IRs possess low toxicity and favorable customer experiences in use (eg, cosmetically pleasant, naturally occurring). This review summarizes the currently available Environmental Protection Agency-registered IRs, including their origins, mechanisms of action, side effect profiles, and available formulations. This review will enable the clinician to select the best IR option to meet a patient's needs and provide the greatest protection from arthropod bites and their sequelae.

Monitoring of serum 25-(OH)D level in infants and its correlation with bone density and development.

Serum 25-hydroxyvitamin D (25(OH)D) is an indicator of nutritional status in the body. Vitamin D (VD) is important for promoting calcium and phosphorus absorption and bone health. This work investigated the correlation between 25(OH)D level and bone density and bone development in infants. the bone density in 150 infants aged 0 to 3 years was measured by ultrasound. Based on the values of bone density, the infants were grouped into a normal (N) group (n = 95) and an abnormal (ABN) group (n = 55). At the same time, serum 25(OH)D, calcium, phosphorus, alkaline phosphatase (ALP), and parathyroid hormone (PTH) levels were detected to analyze their correlations. 25(OH)D, calcium, and phosphorus levels in the ABN group were greatly decreased, while ALP and PTH levels were increased obviously, all presenting remarkable differences with those in the N group (P<0.05). 25(OH)D was positively linked with bone density (r=0.918, P<0.01), calcium level (r=0.316, P<0.05) and phosphorus level (r=0.209, P<0.05) but showed negative associations with ALP level (r=-0.428, P<0.01) and PTH level (r=-0.327, P<0.05). elevating 25(OH)D was crucial in reducing the incidence of abnormal bone density, bettering bone metabolism, and improving the bone health of infants.

Biomonitoring Equivalents for N,N-Diethyl-meta-toluamide (DEET).

N,N-Diethyl-meta-toluamide (DEET) is widely used as an effective mosquito and tick repellent. DEET is absorbed systemically after applications to skin. Once absorbed, DEET is rapidly metabolized with the predominant metabolite being m-dimethylaminocarbonyl benzoic acid (DBA). DEET and metabolites are predominantly excreted in urine after being absorbed systemically. Exposures to DEET are typically biomonitored via measures of DEET and DBA in urine. In this evaluation, we review available health-based risk assessments and toxicological reference values (TRVs) for DEET and derive Biomonitoring Equivalent (BE) values for interpretation of population biomonitoring data. BEs were derived based on existing TRVs derived by Health Canada, yielding 38 and 23 mg/L DBA in urine for adults and 57 and 34 mg/L DBA in urine in children for the acute oral and intermediate dermal TRVs, respectively. The BEs for unchanged DEET in urine are 21 and 12 mg/L in adults and 4.5 and 2.7 mg/L in children for the acute oral and intermediate dermal TRVs. The BE values derived in this manuscript can serve as a guide to help public health officials and regulators interpret population based DEET biomonitoring data in a public health risk context.

The olfactory basis of orchid pollination by mosquitoes.

Mosquitoes are important vectors of disease and require sources of carbohydrates for reproduction and survival. Unlike host-related behaviors of mosquitoes, comparatively less is understood about the mechanisms involved in nectar-feeding decisions, or how this sensory information is processed in the mosquito brain. Here we show that Aedes spp. mosquitoes, including Aedes aegypti, are effective pollinators of the Platanthera obtusata orchid, and demonstrate this mutualism is mediated by the orchid's scent and the balance of excitation and inhibition in the mosquito's antennal lobe (AL). The P. obtusata orchid emits an attractive, nonanal-rich scent, whereas related Platanthera species-not visited by mosquitoes-emit scents dominated by lilac aldehyde. Calcium imaging experiments in the mosquito AL revealed that nonanal and lilac aldehyde each respectively activate the LC2 and AM2 glomerulus, and remarkably, the AM2 glomerulus is also sensitive to N,N-diethyl-meta-toluamide (DEET), a mosquito repellent. Lateral inhibition between these 2 glomeruli reflects the level of attraction to the orchid scents. Whereas the enriched nonanal scent of P. obtusata activates the LC2 and suppresses AM2, the high level of lilac aldehyde in the other orchid scents inverts this pattern of glomerular activity, and behavioral attraction is lost. These results demonstrate the ecological importance of mosquitoes beyond operating as disease vectors and open the door toward understanding the neural basis of mosquito nectar-seeking behaviors.

Challenges encountered by natural repellents: Since obtaining until the final product.

Vector-borne diseases, particularly the arboviruses dengue, Zika, chikungunya, and yellow fever caused by the Aedes aegypti mosquito, have been driving the use of repellents worldwide. The most representative synthetic repellent, DEET stands out as the market's oldest and most efficient repellent. It is considered a reference standard but presents considerable toxicity, not recommended for children up to 6 months old and pregnant women. For this reason, alternatives have been sought, and natural repellents derived mainly from essential oils have been studied, highlighting the essential oils of lemon (Corymbia citriodora), citronella (Cympobogon sp.), Andiroba (Carapa guianensis). However, the development and commercialization of products containing natural repellents are significantly lower when compared to DEET and other synthetic repellents. In order to understand the reasons, aspects related to safety, mechanism of action, efficacy as well development and complexity of the products were evaluated. It is concluded that, as for safety, there is lacking information in the literature regarding the effects on non-target organisms and robust toxicity data. The mechanism of action is based on theories, with less information on the exact mode of action, molecular targets, and interaction with the olfactory and taste receptors of insects. Despite being a current trend to search for actives from natural sources highly present in essential oils, however they reduced action time because due to rapid evaporation after application to the skin, thus requiring repellent vehicles. The development and complexity related to these products bring challenging aspects, beginning on the plant cultivation and extraction processes to produce essential oils with a more homogeneous chemical composition towards the formulation stabilization processes due to fast evaporation and short action time, with the use of pharmaceutical technology such as encapsulation techniques.

Transfluthrin enhances odorant receptor-mediated spatial repellency in Aedes aegypti.

Mosquito-borne diseases are an increasing global health challenge and vector-based interventions remain the most important methods for reducing the public health burden of most mosquito-borne diseases. Transfluthrin, a volatile pyrethroid insecticide, is used widely and effectively as an insecticide and as a repellent. In a recent study, we showed that at very low concentrations transfluthrin repels Aedes aegypti mosquitoes in a hand-in-cage assay without detectable stimulation of antennal olfactory responses. Furthermore, activation of sodium channels by transfluthrin enhances repellency by DEET, which has been reported to repel mosquitoes via odorant receptor (Or)-dependent and Or-independent mechanisms. However, whether activation of sodium channels by transfluthrin can serve as a general mechanism for synergizing the activities of other repellents remain unknown. In this study, we found that, in hand-in-cage assay, transfluthrin enhanced repellency by geranyl acetate and (E)-ß-farnesene, which activate AaOr31. Such enhancement was not observed in AaOr31-knockout mosquitoes and dampened in a pyrethroid-resistant strain carrying two sodium channel mutations, which reduce the action of transfluthrin on sodium channels. In addition, transfluthrin also enhanced repellency by (-)-borneol, (±)-citronellal, camphor, and eucalyptol, which activate Or-mediated repellency. Our study has uncovered the ability of transfluthrin to enhance the repellency to a variety of mosquito repellents, beyond DEET, and provided experimental support for the emerging paradigm of synergistic interactions between repellency mediated by sodium channel activation and Or activation. These findings have potential implications in the development of more effective mosquito repellent mixtures.

[The correlation between nutritional status, body composition, and symptoms in Parkinson's disease patients].

Objective: To investigate the relationship between nutritional status, body composition and symptoms of Parkinson's disease (PD). Methods: The study was conducted among 102 PD patients between November 2020 to April 2021 in the outpatient and inpatient of Neurology department of the Second Affiliated Hospital of Suzhou University. Basic data, such as age, gender, course of disease and Hoehn-Yahr (H-Y) stage of the patients, were collected. In the "open" state, the Unified Parkinson's Disease Rating Scale (UPDRS) and other scales were used to evaluate the clinical symptoms of PD patients. The nutritional status of PD patients was evaluated by the micro-nutrition rating Scale (MNA), and the body composition data of PD patients was measured by the body composition analyzer.The patients were divided into three groups according to the MNA: well-nourished group(45 cases), malnutrition-risk group (43 cases)and malnutrition group(14 cases). Body composition, laboratory tests, bone density, and symptoms of PD were compared among the three groups. Results: Among 102 PD patients, 48 were males (47.1%) and 54 were females (52.9%), aged 42-86 (66±9) years old. Compared with the malnutrition-risk group, lymphocytes in the malnutrition group [(1.45±0.48) × 109/L vs (1.76±0.43)×109/L], uric acid [(227±54) µmol/L vs (282±63) µmol/L] were all low, and the difference was statistically significant (all P<0.05). Compared with the well-nourished group, hemoglobin [(125±17) g/L vs (136±14) g/L], prealbumin [(0.23±0.05) g/L vs (0.27±0.06) g/L], and uric acid [(227±54) µmol/L vs (312±76) µmol/L] and Retinol binding protein [(33±7) mg/L were lower than (39±10) mg/L] in the malnutrition group, the difference was statistically significant (P<0.05). Except for the visceral fat area and body fat percentages, there was no significant difference in body composition indexes and bone mineral density T-value between the malnutrition-risk and malnutrition groups (P<0.05). Compared with the well-nourished group, the body fat [(16±6) kg vs (20±6) kg], the percentage of body fat [(26±9)% vs (29±7)%], the waist-hip ratio [(0.86±0.05) vs (0.89±0.05)], and the upper arm circumference [(29.00±2.59) cm vs (30.74±2.75) cm] in the malnutrition risk group were lower, and the difference was statistically significant (P<0.05). There were statistically significant differences in all body composition indicators and T-values of bone density between the malnourished and well nourished groups (all P<0.05). The MNA score of PD patients was negatively correlated with UPDRS Ⅰ, UPDRS Ⅱ, and UPDRS Ⅳ scores (r=-0.347, -0.364, -0.303, all P<0.05); Body composition indicators: total body water and inorganic salts were negatively correlated with UPDRS-I (r=-0.206, -0.223, all P<0.05), and body fat was negatively correlated with levodopa Equivalent dose (LED) (r=-0.209, P<0.05) Conclusions: The malnutrition risk and incidence of malnutrition in PD patients are high; lymphocyte, uric acid, hemoglobin, Retinol binding protein, body composition and bone mineral density were low in malnutrition group. MNA score of PD patients was negatively correlated with Parkinson's disease symptoms.

Volatile allosteric antagonists of mosquito odorant receptors inhibit human-host attraction.

Odorant-dependent behaviors in insects are triggered by the binding of odorant ligands to the variable subunits of heteromeric olfactory receptors. Previous studies have shown, however, that specific odor binding to ORco, the common subunit of odorant receptor heteromers, may allosterically alter olfactory receptor function and profoundly affect subsequent behavioral responses. Using an insect cell-based screening platform, we identified and characterized several antagonists of the odorant receptor coreceptor of the African malaria vector Anopheles gambiae (AgamORco) in a small collection of natural volatile organic compounds. Because some of the identified antagonists were previously shown to strongly repel Anopheles and Culex mosquitoes, we examined the bioactivities of the identified antagonists against Aedes, the third major genus of the Culicidae family. The tested antagonists inhibited the function of Ae. aegypti ORco ex vivo and repelled adult Asian tiger mosquitoes (Ae. albopictus). Binary mixtures of specific antagonists elicited higher repellency than single antagonists, and binding competition assays suggested that this enhanced repellence is due to antagonist interaction with distinct ORco sites. Our results also suggest that the enhanced mosquito repellency by antagonist mixtures is due to additive rather than synergistic effects of the specific antagonist combinations on ORco function. Taken together, these findings provide novel insights concerning the molecular aspects of odorant receptor function. Moreover, our results demonstrate that a simple screening assay may be used for the identification of allosteric modifiers of olfactory-driven behaviors capable of providing enhanced personal protection against multiple mosquito-borne infectious diseases.

Di-Pyridine-Containing Macrocyclic Triamide Fe(II) and Ni(II) Complexes as ParaCEST Agents.

Fe(II) and Ni(II) paraCEST contrast agents containing the di-pyridine macrocyclic ligand 2,2',2″-(3,7,10-triaza-1,5(2,6)-dipyridinacycloundecaphane-3,7,10-triyl)triacetamide (DETA) are reported here. Both [Fe(DETA)]2+ and [Ni(DETA)]2+ complexes were structurally characterized. Crystallographic data revealed the seven-coordinated distorted pentagonal bipyramidal geometry of the [Fe(DETA)]·(BF4)2·MeCN complex with five coordinated nitrogen atoms from the macrocyclic ring and two coordinated oxygen atoms from two amide pendant arms. The [Ni(DETA)]·Cl2·2H2O complex was six-coordinated in nature with a distorted octahedral geometry. Four coordinated nitrogen atoms were from the macrocyclic ring, and two coordinated oxygen atoms were from two amide pendant arms. [Fe(DETA)]2+ exhibited well-resolved sharp proton resonances, whereas very broad proton resonances were observed in the case of [Ni(DETA)]2+ due to the long electronic relaxation times. The CEST peaks for the [Fe(DETA)]2+ complex showed one highly downfield-shifted and intense peak at 84 ppm with another shifted but less intense peak at 28 ppm with good CEST contrast efficiency at body temperature, whereas [Ni(DETA)]2+ showed only one highly shifted intense peak at 78 ppm from the bulk water protons. Potentiometric titrations were performed to determine the protonation constants of the ligand and the thermodynamic stability constant of the [M(DETA)]2+ (M = Fe, Co, Ni, Cu, Zn) species at 25.0 °C and I = 0.15 mol·L-1 NaClO4. Metal exchange studies confirmed the stability of the complexes in acidic medium in the presence of physiologically relevant anions and an equimolar concentration of Zn(II) ions.

Scavenging of hexavalent chromium from aqueous solution by Macadamia nutshell biomass modified with diethylenetriamine and maleic anhydride.

Based on the premise that aqueous anions of hexavalent chromium (Cr(VI)) are capable of electrostatic interaction with cationic and polar active sites, acid-washed Madacamia nutshell biomass was sequentially treated with diethylenetriamine (DETA) and maleic anhydride (MA) to graft poly(diethylenetriamine-co-maleic anhydride). By displaying a new peak at 1685 cm-1 ascribed to amide CO stretching vibrations, Fourier transform infrared spectroscopy highlighted the formation of amide groups through reaction of DETA with carboxyl groups on the biomass surface. Scanning electron microscopic images of the MA-modified biomass displayed polymeric growths attributed to copolymerization of DETA with MA. The polar and ionizable amide and amine groups of the grafted copolymer endowed the adsorbent with Cr(VI) removal capabilities over a wide pH range demonstrated by removal efficiencies between 70.9% and 81.7% in the pH 1.6 to pH 10.0 range for the treatment of 20 mL solutions containing 100 mg L-1 Cr(VI) with 200 mg of adsorbent. Conformity of the adsorption isotherm data to the Freundlich model revealed the heterogeneous nature of the adsorbent surface, which comprised a variety of functional groups capable of interaction with Cr(VI) species in solution. The Sips isotherm model provided the best fit to the equilibrium experimental data, and the adsorption capacity was 779.1 mg g-1 at pH 1.6, room temperature and an adsorbent dosage of 5.0 g L-1. The findings indicate that Cr(VI) adsorption onto diethylenetriamine and maleic anhydride modified Madacamia nutshell biomass is a promising option for Cr(VI) removal from aqueous solutions.

A rapid method for screening mosquito repellents on Anopheles pseudopunctipennis and Aedes aegypti.

One of the main vectors for malaria in Latin America is Anopheles pseudopunctipennis (Theobald), whereas Aedes aegypti (L.) is the primary vector of dengue, yellow fever, Zika and chikungunya viruses. The use of repellents is recommended as a personal protection method against these mosquitoes. However there are very few studies evaluating the effect of repellents on An. pseudopunctipennis. The use of a Petri dish to study repellence has been applied by several authors on flies, cockroaches, kissing bugs and mosquitoes, being a valuable technique for species that are difficult to breed under laboratory conditions, such as An. pseudopunctipennis. In the present study, we evaluated the repellence of the essential oil of the Eucalyptus nitens (Shining gum), its main component (1,8-cineole) and the commercial repellent DEET on An. pseudopunctipennis and Ae. aegypti adult females using the plaque repellency method coupled to EthoVision XT10.1 video-tracking software. Repellent effect and locomotor activity were studied through a repellence index (RI) together with an axis constructed from the behavioural variables obtained using the tracking software. DEET repellent effect was observed at 0.01 mg/mL for Ae. aegypti and 0.01 and 0.1 mg/mL for An. pseudopunctipennis. In addition, the essential oil showed significant repellence at 1 and 10 mg/mL for Ae. aegypti, and 1, 5, 10 and 25 mg/mL for An. pseudopunctipennis. Neither of these species were repelled at any concentration of 1,8-cineole. This is the first study that evaluates these compounds on An. pseudopunctipennis females and quantifies their effects on the activity of both species.

Microencapsulation of DEET in Solid Lipid Microparticles: production, characterization and safety evaluation.

DEET is considered the gold standard for insect repellent products. However, it behaves as a strong skin permeant. DEET was encapsulated in Solid Lipid Microparticles (SLM) and characterized in terms of morphology, particle size, cytotoxicity and ex vivo permeation. The particles exhibited micrometric size with a spherical shape. In addition, we developed and validated an analytical method for DEET quantification by high performance liquid chromatography (HPLC), which was selective, linear, precise, accurate and robust. The toxicity test in cell culture of keratinocytes, fibroblasts and macrophages showed that the formulation did not present cytotoxicity. The SLM were able to decrease the skin permeation of DEET in relation to the free active in ethanol with gain in the safe. Microparticles were able to increase the skin retention of DEET, which can contribute to extend the time of repellent action. The results showed that Solid Lipid Microparticles are safe and promising topical formulation to insect bite prevention.

Antifungal, Repellency, and Insecticidal Activities of Cymbopogon distans and Ruta graveolens Essential Oils and Their Main Chemical Constituents.

Essential oils produced by Cymbopogon distans and Ruta graveolens with a similar Chinese name could be explained as book fragrance in the Chinese idiom 'shu xiang men di', namely, a wealthy intellectual family according to ancient Chinese. Therefore, volatile oils from these two plants and their main compounds were tested to explore their antifungal, repellent, and insecticide actions. In this study, the essential olis (EO) of C. distans exhibited significant antifungal activity against Rhizopus stolonifera (97 %), Mucor racemosus (97 %), and Trichoderma viride (84 %); its main compounds exhibit interesting activity, such as methyleugenol (87 %) and elemicine (85 %) against T. viride and butyl hydroxytoluene against M. racemosus (90 %) and R. stolonifera (95 %). The EO of R. graveolens and other major chemical constituents showed weak inhibitory effects against other fungi (Aspergillus flavus and Fusarium oxysporum). Then, EO (C. distans and R. graveolens) and its main compounds exhibited obvious repellent activity (more than 85 %) at a concentration of 16 nL/cm2 , which was consistent with the repellency of the positive control (DEET). In terms of insecticidal activity, the mortality of C. distans volatile oil against R. padi (56.4 %) was lower than that of R. graveolens volatile oil (92.4 %), and all compounds showed weak lethal effects. These results provide a natural substance for controlling fungi and insects when storing books and that can be used as a biological pesticide for industrial production. Through our study, the book fragrance in the Chinese idiom 'shu xiang men di' was speculated to be the EO odor of C. distans.

Discovery of the Key Mutation Site Influencing the Thermostability of Thermomyces lanuginosus Lipase by Rosetta Design Programs.

Lipases are remarkable biocatalysts and are broadly applied in many industry fields because of their versatile catalytic capabilities. Considering the harsh biotechnological treatment of industrial processes, the activities of lipase products are required to be maintained under extreme conditions. In our current study, Gibbs free energy calculations were performed to predict potent thermostable Thermomyces lanuginosus lipase (TLL) variants by Rosetta design programs. The calculating results suggest that engineering on R209 may greatly influence TLL thermostability. Accordingly, ten TLL mutants substituted R209 were generated and verified. We demonstrate that three out of ten mutants (R209H, R209M, and R209I) exhibit increased optimum reaction temperatures, melting temperatures, and thermal tolerances. Based on molecular dynamics simulation analysis, we show that the stable hydrogen bonding interaction between H198 and N247 stabilizes the local configuration of the 250-loop in the three R209 mutants, which may further contribute to higher rigidity and improved enzymatic thermostability. Our study provides novel insights into a single residue, R209, and the 250-loop, which were reported for the first time in modulating the thermostability of TLL. Additionally, the resultant R209 variants generated in this study might be promising candidates for future-industrial applications.