Molecular and Functional Characterization of Three General Odorant-Binding Protein 2 Genes in Cydia pomonella (Lepidoptera: Tortricidae).

General odorant-binding proteins (GOBPs) play a crucial role in the detection of host plant volatiles and pheromones by lepidopterans. Previous studies identified two duplications in the GOBP2 gene in Cydia pomonella. In this study, we employed qRT-PCR, protein purification, and fluorescence competitive binding assays to investigate the functions of three GOBP2 genes in C. pomonella. Our findings reveal that CpomGOBP2a and CpomGOBP2b are specifically highly expressed in antennae, while CpomGOBP2c exhibits high specific expression in wings, suggesting a potential divergence in their functions. Recombinant proteins of CpomGOBP2a, CpomGOBP2b, and CpomGOBP2c were successfully expressed and purified, enabling an in-depth exploration of their functions. Competitive binding assays with 20 host plant volatiles and the sex pheromone (codlemone) demonstrated that CpomGOBP2a exhibits strong binding to four compounds, namely butyl octanoate, ethyl (2E,4Z)-deca-2,4-dienoate (pear ester), codlemone, and geranylacetone, with corresponding dissolution constants (Ki) of 8.59993 µM, 9.14704 µM, 22.66298 µM, and 22.86923 µM, respectively. CpomGOBP2b showed specific binding to pear ester (Ki = 17.37481 µM), while CpomGOBP2c did not exhibit binding to any tested compounds. In conclusion, our results indicate a functional divergence among CpomGOBP2a, CpomGOBP2b, and CpomGOBP2c. These findings contribute valuable insights for the development of novel prevention and control technologies and enhance our understanding of the evolutionary mechanisms of olfactory genes in C. pomonella.

Characterization of the off-flavor from Pichia pastoris GS115 during the overexpression of an α-l-rhamnosidase.

The off-flavor of Pichia pastoris strains is a negative characteristic of proteins overexpressed with this yeast. In the present study, P. pastoris GS115 overexpressing an α-l-rhamnosidase was taken as the example to characterize the off-flavor via sensory evaluation, gas chromatography-mass spectrometer, gas chromatography-olfaction, and omission test. The result showed that the off-flavor was due to the strong sweaty note, and moderate metallic and plastic notes. Four volatile compounds, that is, tetramethylpyrazine, 2,4-di-tert-butylphenol, isovaleric acid, and 2-methylbutyric acid, were identified to be major contributors to the sweaty note. Dodecanol and 2-acetylbutyrolactone were identified to be contributors to the metallic and plastic notes, respectively. It is the first study on the off-flavor of P. pastoris strains, helping understand metabolites with off-flavor of this yeast. Interestingly, it is the first study illustrating 2-acetylbutyrolactone and dodecanol with plastic and metallic notes, providing new information about the aromatic contributors of biological products. IMPORTANCE: The methylotrophic yeast Pichia pastoris is an important host for the industrial expression of functional proteins. In our previous studies, P. pastoris strains have been sniffed with a strong off-flavor during the overexpression of various functional proteins, limiting the application of these proteins. Although many yeast strains have been reported with off-flavor, no attention has been paid to characterize the off-flavor in P. pastoris so far. Considering that P. pastoris has advantages over other established expression systems of functional proteins, it is of interest to identify the compounds with off-flavor synthesized in the overexpression of functional proteins with P. pastoris strains. In this study, the off-flavor synthesized from P. pastoris GS115 was characterized during the overexpression of an α-l-rhamnosidase, which helps understand the aromatic metabolites with off-flavor of P. pastoris strains. In addition, 2-acetylbutyrolactone and dodecanol were newly revealed with plastic and metallic notes, enriching the aromatic contributors of biological products. Thus, this study is important for understanding the metabolites with off-flavor of P. pastoris strains and other organisms, providing important knowledge to improve the flavor of products yielding with P. pastoris strains and other organisms. ONE-SENTENCE SUMMARY: Characterize the sensory and chemical profile of the off-flavor produced by one strain of P. pastoris in vitro.

Chemical composition and antibacterial effects of Etlingera elatior (Jack) R.M. Smith against Staphylococcus aureus efflux pumps.

Multidrug resistance is a significant health problem worldwide, with increasing mortality rates, especially in the last few years. In this context, a consistent effort has been made to discover new antibacterial agents, and evidence points to natural products as the most promising source of bioactive compounds. This research aimed to characterize the antibacterial effect of the essential oil of Etlingera elatior (EOEE) and its major constituents against efflux pump-carrying Staphylococcus aureus strains. The essential oil was extracted from fresh inflorescences by hydrodistillation. Chemical analysis was performed using gas chromatography coupled to mass spectrometry (GC-MS) and gas chromatography equipped with a flame ionization detector (GC-FID). The strains RN-4220, 1199B, IS-58, and 1199 of S. aureus were used to evaluate the antibacterial activity and the inhibition of efflux pumps. A total of 23 compounds were identified, including dodecanal and 1-dodecanol as major compounds. EOEE and dodecanal showed weak activity against the strains, while 1-dodecanol inhibited bacterial growth at low concentrations, indicating strong antibacterial activity. In addition, this compound potentiated the activity of norfloxacin against S. aureus 1199. In conclusion, 1-dodecanol was identified as the most effective compound of EOEE, showing significant potential to be used in antibacterial drug development.

Delineation of the CYP505E subfamily of fungal self-sufficient in-chain hydroxylating cytochrome P450 monooxygenases.

Cytochrome P450 monooxygenases (CYP450s) are abundant in eukaryotes, specifically in plants and fungi where they play important roles in the synthesis and degradation of secondary metabolites. In eukaryotes, the best studied "self-sufficient" CYP450s, with a fused redox partner, belong to the CYP505 family. Members of the CYP505 family are generally considered sub-terminal fatty acid hydroxylases. CYP505E3 from Aspergillus terreus, however, gives remarkable in-chain hydroxylation at the ω-7 position of C10 to C16 alkanes and C12 and C14 fatty alcohols. Because CYP505E3 is a promising catalyst for the synthesis of δ-dodecalactone, we set out to delineate the unique ω-7 hydroxylase activity of CYP505E3. CYP505E3 and six additional CYP505Es as well as four closely related CYP505s from four different subfamilies were expressed in Pichia pastoris. Only the CYP505Es, sharing more than 70% amino acid identity, displayed significant ω-7 hydroxylase activity toward 1-dodecanol, dodecanoic acid, and tetradecanoic acid giving products that can readily be converted to δ-dodecalactone. Concentrations of δ-dodecalactone, directly extracted from dodecanoic acid biotransformations, were higher than previously obtained with E. coli. Searches of the UniProt and NCBI databases yielded a total of only 23 unique CYP505Es, all from the Aspergillaceae. Given that CYP505Es with this remarkable activity occur in only a few Aspergillus and Penicillium spp., we further explored the genetic environments in which they occur. These were found to be very distinct environments which include a specific ABC transporter but could not be linked to apparent secondary metabolite gene clusters. KEY POINTS: • Identified CYP505Es share > 70% amino acid identity. • CYP505Es hydroxylate 1-dodecanol, dodecanoic, and tetradecanoic acid at ω-7 position. • CYP505E genes occur in Aspergillus and Penicillium spp. near an ABC transporter.

Immunomodulatory efficacy of Cousinia thomsonii C.B. Clarke in ameliorating inflammatory cascade expressions.

ETHNOPHARMACOLOGICAL RELEVANCE: Cousinia thomsonii is traditionally known for treating various diseases including joint pain, swelling, body ache, asthma, dermatitis, cough and arthritis. AIM OF THE STUDY: This study employs lipopolysaccharide induced inflammatory wistar-rat model to evaluate efficacy of Cousinia thomsonii active-extracts on the expression of crucial inflammatory markers viz. iNOS, PPAR-γ, Rel-A, COX-2 and serum analysis of CRP. MATERIALS AND METHODS: Methanol and aqueous extracts were administered orally at 25, 50, 100 mg/kg doses for 21 days. Serum was collected on 22nd day and rats were sacrificed to extract paw tissues. Dexamethasone (0.5 mg/kg) served as positive control. Immunoblotting and qPCR was used for expression analysis of iNOS, PPAR-γ, Rel-A, COX-2 respectively. ELISA was employed for evaluating CRP levels. Discovery-studio and Auto-Dock-Vina were used to check docking interactions of various identified compounds. RESULTS: Both extracts caused dose-dependent decline in iNOS, Rel-A, COX-2 and CRP levels, while there was a dose-dependent increase in PPAR-γ expression. Methanol extract dominated immunomodulatory potential as compared with the aqueous extract. The results of the GCMS revealed the presence of ten compounds. Some of these compounds include 1-Octacosanol, Ethyl Linoleate, 1-Heptacosanol, 1-Hexadecanol, 1-Dodecanol and Behenic alcohol having strong anti-inflammatory, antimicrobial, anti-acne and anti-viral activities. Molecular Docking scores were calculated between each target protein and selected compounds. The best affinity/interactions were observed between 1-Octacosanol towards iNOS, PPAR-γ, Rel-A, COX-2 and CRP with binding energy of -10.4, -11.1, -8.6, -9.9 and -7.9 (kcal/mol) respectively. These compounds may act as strong inhibitors for iNOS, Rel-A, COX-2 and CRP or as agonists for PPAR-γ; thereby inducing anti-inflammatory/immuno-modulatory activities. CONCLUSIONS: The results indicate that Cousinia thomsonii contains therapeutically active compounds and thus could serve as potential therapeutic regimen against diverse inflammatory diseases.

Tromethamine and dodecanol appear to be the major secondary metabolites of Streptomyces decoyicus M.

An isolate of Streptomyces decoyicus M* (code of the isolate) was identified by the sequencing of 16S rRNA gene. It was grown on solid media and secondary metabolites were extracted with n-butanol. The extract was dried and run in a sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE, 10%). Two main bands obtained were sliced and the metabolites were regained in n-butanol. These two samples were then identified by gas-chromatography-mass spectrometry (GC-MS), and Fourier-transform infrared spectroscopy (FT-IR). The results demonstrated that tromethamine- and 1-dodecanol were the main constituents (band 1: 61% and 17.7%; band 2: 41% and 54%, respectively). This finding maintained that the isolate of Streptomyces decoyicus produced high amounts tromethamine- and 1-dodecanol under the conditions investigated.

Air-assisted in situ deep eutectic solvent decomposition followed by the solidification of floating organic droplets-liquid-liquid microextraction method for extraction of azole antifungal drugs in biological samples prior to high-performance liquid chromatography.

A free dispersive method, air-assisted in situ deep eutectic solvent decomposition followed by the solidification of floating organic droplets liquid-liquid microextraction was indicated in this study. This technique was utilized to simultaneously ascertain some azole antifungal drugs prior to high-performance liquid chromatography. In this research, a quasi-hydrophobic deep eutectic solvent was formed from tetrabutylammonium bromide and 1-dodecanol as an organic solvent at a 1:2 molar ratio. The synthesized deep decomposition in the sample solution caused in situ dispersion of extraction solvent and analytes. Air-assisted enhanced a dispersion condition in the sample solution. 1-Dodecanol as a green option was replaced with typical extraction solvents providing the advantages of a suitable freezing point near room temperature and low density. The effect of important analytical parameters on the extraction recovery of analytes was assessed. Under these optimal conditions, the limits of detection and the limits of quantitation determined were in the range of 0.5-2.8 and 1.5-9 µg/L, for water, urine and plasma samples, respectively. The intra-day and inter-day relative standard deviations (n = 5) were calculated to be 2.9-4.6 and 4.2-8.9%, respectively. The results represented the effectiveness of the developed method for the extraction and determination of analytes in biological samples.

Structural insights into the discrepant synergistic activity of Codlemone and (Z)-8-dodecenol towards Grapholita molesta pheromones.

BACKGROUND: Insect pheromone synergists have been widely used to produce potent pheromone products for environment-friendly pest control. Codlemone (Cod) and (Z)-8-dodecenol (Dod) are two major Grapholita molesta pheromone synergists, with Cod having greater synergism and affinity for G. molesta pheromone binding protein 2 (GmolPBP2). Uncovering structural information key to the different binding affinity of Cod and Dod to GmolPBP2 would gain insights into what causes their synergy activity discrepancy. RESULTS: Binding modes of the two synergists in the binding pocket of GmolPBP2 were analyzed and compared by molecular dynamics-based approaches. Although Cod and Dod were stabilized in a similar hydrophobic pocket, their interaction details with GmolPBP2 were divergent due to the extra double bond (C10═C11) in Cod. The C10═C11 improved the hydrophobic interactions of Cod with around residues. Such hydrophobic interaction improvement was also reflected in the raised importance of Phe11 in the GmolPBP2-Cod interaction. Not only that, the increased hydrophobic forces introduced by the C10═C11 changed the CH2-OH orientation in the GmolPBP2-Cod complex, which improved the H-bond interaction. Electrostatic complementarity analysis further indicated the positive role of C10═C11 in optimizing GmolPBP2-Cod interaction. CONCLUSION: The C10═C11 is thought to contribute greatly to Cod's stronger synergy as a group key to the higher GmolPBP2-affinity, based on which the improvement directions for Cod and Dod were addressed as well. Our findings will aid in the development and optimization of more effective pheromone synergists, resulting in more effective pheromone-based pest management.

Structure-activity relationships of antifungal phenylpropanoid derivatives and their synergy with n-dodecanol and fluconazole.

trans-Anethole (anethole) is a phenylpropanoid; with other drugs, it exhibits synergistic activity against several fungi and is expected to be used in new therapies that cause fewer patient side effects. However, the detailed substructure(s) of the molecule responsible for this synergy has not been fully elucidated. We investigated the structure-activity relationships of phenylpropanoids and related derivatives, with particular attention on the methoxy group and the double bond of the propenyl group in anethole, as well as the length of the p-alkyl chain in p-alkylanisoles. Antifungal potency was largely related to p-alkyl chain length and the methoxy group of anethole, but not to the double bond of its propenyl group. Production of reactive oxygen species also played a role in these fungicidal activities. Inhibition of drug efflux was associated with the length of the p-alkyl chain and the double bond of the propenyl group in anethole, but not with the methoxy group. Although a desirable synergy was observed between n-dodecanol and anethole or p-alkylanisoles with a length of C2-C6 in alkyl chains, it cannot be explained away as being solely due to the inhibition of drug efflux. Similar results were obtained when phenylpropanoid derivatives were combined with fluconazole against Candida albicans.

Green Chemistry Production of Codlemone, the Sex Pheromone of the Codling Moth (Cydia pomonella), by Metabolic Engineering of the Oilseed Crop Camelina (Camelina sativa).

Synthetic pheromones have been used for pest control over several decades. The conventional synthesis of di-unsaturated pheromone compounds is usually complex and costly. Camelina (Camelina sativa) has emerged as an ideal, non-food biotech oilseed platform for production of oils with modified fatty acid compositions. We used Camelina as a plant factory to produce mono- and di-unsaturated C12 chain length moth sex pheromone precursors, (E)-9-dodecenoic acid and (E,E)-8,10-dodecadienoic acid, by introducing a fatty acyl-ACP thioesterase FatB gene UcTE from California bay laurel (Umbellularia californica) and a bifunctional ∆9 desaturase gene Cpo_CPRQ from the codling moth, Cydia pomonella. Different transgene combinations were investigated for increasing pheromone precursor yield. The most productive Camelina line was engineered with a vector that contained one copy of UcTE and the viral suppressor protein encoding P19 transgenes and three copies of Cpo_CPRQ transgene. The T2 generation of this line produced 9.4% of (E)-9-dodecenoic acid and 5.5% of (E,E)-8,10-dodecadienoic acid of the total fatty acids, and seeds were selected to advance top-performing lines to homozygosity. In the T4 generation, production levels of (E)-9-dodecenoic acid and (E,E)-8,10-dodecadienoic acid remained stable. The diene acid together with other seed fatty acids were converted into corresponding alcohols, and the bioactivity of the plant-derived codlemone was confirmed by GC-EAD and a flight tunnel assay. Trapping in orchards and home gardens confirmed significant and specific attraction of C. pomonella males to the plant-derived codlemone.

Dodecanol, metabolite of entomopathogenic fungus Conidiobolus coronatus, affects fatty acid composition and cellular immunity of Galleria mellonella and Calliphora vicina.

One group of promising pest control agents are the entomopathogenic fungi; one such example is Conidiobolus coronatus, which produces a range of metabolites. Our present findings reveal for the first time that C. coronatus also produces dodecanol, a compound widely used to make surfactants and pharmaceuticals, and enhance flavors in food. The main aim of the study was to determine the influence of dodecanol on insect defense systems, i.e. cuticular lipid composition and the condition of insect immunocompetent cells; hence, its effect was examined in detail on two species differing in susceptibility to fungal infection: Galleria mellonella and Calliphora vicina. Dodecanol treatment elicited significant quantitative and qualitative differences in cuticular free fatty acid (FFA) profiles between the species, based on gas chromatography analysis with mass spectrometry (GC/MS), and had a negative effect on G. mellonella and C. vicina hemocytes and a Sf9 cell line in vitro: after 48 h, almost all the cells were completely disintegrated. The metabolite had a negative effect on the insect defense system, suggesting that it could play an important role during C. coronatus infection. Its high insecticidal activity and lack of toxicity towards vertebrates suggest it could be an effective insecticide.

Biosynthesis of the Sex Pheromone Component (E,Z)-7,9-Dodecadienyl Acetate in the European Grapevine Moth, Lobesia botrana, Involving ∆11 Desaturation and an Elusive ∆7 Desaturase.

The European grapevine moth, Lobesia botrana, uses (E,Z)-7,9-dodecadienyl acetate as its major sex pheromone component. Through in vivo labeling experiments we demonstrated that the doubly unsaturated pheromone component is produced by ∆11 desaturation of tetradecanoic acid, followed by chain shortening of (Z)-11-tetradecenoic acid to (Z)-9-dodecenoic acid, and subsequently introduction of the second double bond by an unknown ∆7 desaturase, before final reduction and acetylation. By sequencing and analyzing the transcriptome of female pheromone glands of L. botrana, we obtained 41 candidate genes that may be involved in sex pheromone production, including the genes encoding 17 fatty acyl desaturases, 13 fatty acyl reductases, 1 fatty acid synthase, 3 acyl-CoA oxidases, 1 acetyl-CoA carboxylase, 4 fatty acid transport proteins and 2 acyl-CoA binding proteins. A functional assay of desaturase and acyl-CoA oxidase gene candidates in yeast and insect cell (Sf9) heterologous expression systems revealed that Lbo_PPTQ encodes a ∆11 desaturase producing (Z)-11-tetradecenoic acid from tetradecanoic acid. Further, Lbo_31670 and Lbo_49602 encode two acyl-CoA oxidases that may produce (Z)-9-dodecenoic acid by chain shortening (Z)-11-tetradecenoic acid. The gene encoding the enzyme introducing the E7 double bond into (Z)-9-dodecenoic acid remains elusive even though we assayed 17 candidate desaturases in the two heterologous systems.

A New CYP2E1 Inhibitor, 12-Imidazolyl-1-dodecanol, Represents a Potential Treatment for Hepatocellular Carcinoma.

Cytochrome P450 2E1 (CYP2E1) is a key target protein in the development of alcoholic and nonalcoholic fatty liver disease (FLD). The pathophysiological correlate is the massive production of reactive oxygen species. The role of CYP2E1 in the development of hepatocellular carcinoma (HCC), the final complication of FLD, remains controversial. Specifically, CYP2E1 has not yet been defined as a molecular target for HCC therapy. In addition, a CYP2E1-specific drug has not been developed. We have already shown that our newly developed CYP2E1 inhibitor 12-imidazolyl-1-dodecanol (I-ol) was therapeutically effective against alcoholic and nonalcoholic steatohepatitis. In this study, we investigated the effect of I-ol on HCC tumorigenesis and whether I-ol could serve as a possible treatment option for terminal-stage FLD. I-ol exerted a very highly significant antitumour effect against hepatocellular HepG2 cells. Cell viability was reduced in a dose-dependent manner, with only the highest doses causing a cytotoxic effect associated with caspase 3/7 activation. Comparable results were obtained for the model colorectal adenocarcinoma cell line, DLD-1, whose tumorigenesis is also associated with CYP2E1. Transcriptome analyses showed a clear effect of I-ol on apoptosis and cell-cycle regulation, with the increased expression of p27Kip1 being particularly noticeable. These observations were confirmed at the protein level for HepG2 and DLD-1 cells grafted on a chorioallantoic membrane. Cell-cycle analysis showed a complete loss of proliferating cells with a simultaneous increase in S-phase arrest beginning at a threshold dose of 30 µM. I-ol also reduced xenograft tumour growth in nude mice. This antitumour effect was not associated with tumour cachexia. I-ol was not toxic to healthy tissues or organs. This study demonstrates for the first time the therapeutic effect of the specific CYP2E1 inhibitor I-ol on the tumorigenesis of HCC. Our findings imply that I-ol can potentially be applied therapeutically on patients at the final stage of FLD.

Solidified floating organic drop microextraction (SFODME) for the simultaneous analysis of three non-steroidal anti-inflammatory drugs in aqueous samples by HPLC.

In this work, a liquid-liquid microextraction methodology using solidified floating organic drop (SFODME) was combined with liquid chromatography and UV/Vis detection to determine non-steroidal anti-inflammatory drugs (NSAIDs) naproxen (NPX), diclofenac (DCF), and mefenamic acid (MFN) in tap water, surface water, and seawater samples. Parameters that can influence the efficiency of the process were evaluated, such as the type and volume of the extractor and dispersive solvents, effect of pH, agitation type, and ionic strength. The optimized method showed low detection limits (0.09 to 0.25 µg L-1), satisfactory recovery rates (90 to 116%), and enrichment factors in the range between 149 and 199. SFODME showed simplicity, low cost, speed, and high concentration capacity of the analytes under study. Its use in real samples did not demonstrate a matrix effect that would compromise the effectiveness of the method, being possible to apply it successfully in water samples with different characteristics.

A Novel Isolate (S15) of Streptomyces griseobrunneus Produces 1-Dodecanol.

One-dodecanol was identified to be the predominant secondary metabolite of a novel isolate (S15) of Streptomyces griseobrunneus. For its demonstration, secondary metabolite extracts were electrophoresed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). A yellowish unique band was then cut out from the gel and its metabolite content was eluted in n-butanol. GC-MS analysis indicated that more than 93% of the of the elution material were 1-dodecanol. The compound was further characterized by FTIR and 13C NMR analyses. Dendrogram built on the basis of 16S rRNA gene sequence indicated that the isolate S15 was a member of Streptomyces griseobrunneus.

A machine learning Automated Recommendation Tool for synthetic biology.

Synthetic biology allows us to bioengineer cells to synthesize novel valuable molecules such as renewable biofuels or anticancer drugs. However, traditional synthetic biology approaches involve ad-hoc engineering practices, which lead to long development times. Here, we present the Automated Recommendation Tool (ART), a tool that leverages machine learning and probabilistic modeling techniques to guide synthetic biology in a systematic fashion, without the need for a full mechanistic understanding of the biological system. Using sampling-based optimization, ART provides a set of recommended strains to be built in the next engineering cycle, alongside probabilistic predictions of their production levels. We demonstrate the capabilities of ART on simulated data sets, as well as experimental data from real metabolic engineering projects producing renewable biofuels, hoppy flavored beer without hops, fatty acids, and tryptophan. Finally, we discuss the limitations of this approach, and the practical consequences of the underlying assumptions failing.

Structure dynamics reveal key residues essential for the sense of 1-dodecanol by Cydia pomonella pheromone binding protein 2 (CpomPBP2).

BACKGROUND: Cydia pomonella, a worldwide quarantine fruit pest, causes great damage to fruit production every year. Sex pheromone-mediated control of C. pomonella has been widely used. As an indispensable ingredient of commercial sex attractants, 1-dodecanol (Dod) works to synergize the effect of codlemone in attracting male moths of C. pomonella. The interactions between Dod and its transporter protein, C. pomonella pheromone-binding protein 2 (CpomPBP2), provide inspiration for chemical optimizations to improve the synergistic effects of Dod. RESULTS: In this research, molecular simulations and biological verifications were used in combination to uncover key residues in CpomPBP2 essential for sensing Dod. After performing 150 ns molecular dynamics (MD) simulations, the C1-C12 chain of Dod was found to be locked by the van der Waals energy contributed by the hydrophobic residues Phe12, Leu68, and Ile113, whereas the -OH part of Dod was anchored by the H-bond derived from Glu98 and the salt-bridge derived from Arg109. Because of the importance of these two electrostatic interactions, Glu98 and Arg109 were further verified as key residues in determining the binding affinity between Dod and CpomPBP2. In addition, interactions unfavorable to the binding of Dod were described. CONCLUSION: The research detailed the discovery of key residues involved in CpomPBP2-Dod interactions. Our results provide guidance and caution for the prospective discovery, optimization, and design of novel chemicals with a similar or stronger synergistic effect to codlemone in controlling C. pomonella.

Sexual communication of Spodoptera frugiperda from West Africa: Adaptation of an invasive species and implications for pest management.

The pest species Spodoptera frugiperda, which is native to North and South America, has invaded Africa in 2016. The species consists of two strains, the corn-strain and rice-strain, which differ in their sexual communication. When we investigated populations from Benin and Nigeria, consisting of corn-strain and rice-corn-hybrid descendants, we found no strain-specific sexual communication differences. Both genotypes exhibited the same pheromone composition, consisting of around 97% (Z)-9-tetradecenyl acetate (Z9-14:Ac), 2% (Z)-7-dodecenyl acetate (Z7-12:Ac), and 1% (Z)-9-dodecenyl acetate (Z9-12:Ac), they had similar electrophysiological responses, and all mated around three hours into scotophase. However, we found geographic variation between African and American populations. The sex pheromone of African corn-strain and hybrid descendant females was similar to American rice-strain females and showed higher percentages of the male-attracting minor component Z7-12:Ac. In addition, African males exhibited the highest antennal sensitivity towards Z7-12:Ac, while American males showed highest sensitivity towards the major pheromone component Z9-14:Ac. Increasing the production of and response to the critical minor component Z7-12:Ac may reduce communication interference with other African Spodoptera species that share the same major pheromone component. The implications of our results on pheromone-based pest management strategies are discussed.

Deletion of the Golgi Ca2+-ATPase PMR1 gene potentiates antifungal effects of dodecanol that depend on intracellular Ca2+ accumulation in budding yeast.

One strategy for overcoming infectious diseases caused by drug-resistant fungi involves combining drugs rendered inactive by resistance with agents targeting the drug resistance mechanism. The antifungal activity of n-dodecanol disappears as incubation time passes. In Saccharomyces cerevisiae, anethole, a principal component of anise oil, prolongs the transient antifungal effect of dodecanol by downregulating genes of multidrug efflux pumps, mainly PDR5. However, the detailed mechanisms of dodecanol's antifungal action and the anethole-induced prolonged antifungal action of dodecanol are unknown. Screening of S. cerevisiae strains lacking genes related to Ca2+ homeostasis and signaling identified a pmr1Δ strain lacking Golgi Ca2+-ATPase as more sensitive to dodecanol than the parental strain. Dodecanol and the dodecanol + anethole combination significantly increased intracellular Ca2+ levels in both strains, but the mutant failed to clear intracellular Ca2+ accumulation. Further, dodecanol and the drug combination reduced PMR1 expression and did not lead to specific localization of Pmr1p in the parental strain after 4-h treatment. By contrast with the parental strain, dodecanol did not stimulate PDR5 expression in pmr1Δ. Based on these observations, we propose that the antifungal activity of dodecanol is related to intracellular Ca2+ accumulation, possibly dependent on PMR1 function, with anethole enabling Ca2+ accumulation by restricting dodecanol efflux.

The use of micelles to deliver potential hedgehog pathway inhibitor for the treatment of liver fibrosis.

Rationale: Hedgehog (Hh) pathway plays an essential role in liver fibrosis by promoting the proliferation of hepatic stellate cells (HSCs) by enhancing their metabolism via yes-associated protein 1 (YAP1). Despite the presence of several inhibitors, Hh signaling cannot be controlled exclusively due to their poor efficacy and the lack of a suitable delivery system to the injury site. Therefore, it is rationale to develop new potent Hh inhibitors and suitable delivery carriers. Methods: Based on the structure and activity of Hh inhibitor GDC-0449, we replaced its sulfonamide group with two methylpyridine-2yl at amide nitrogen to synthesize MDB5. We compared the Hh pathway inhibition and anti-fibrotic effect of MDB5 with GDC-0449 in vitro. Next, we developed MDB5 loaded micelles using our methoxy poly(ethylene glycol)-blockpoly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol (PEG-PCC-g-DC) copolymer and characterized for physicochemical properties. We evaluated the therapeutic efficacy of MDB5 loaded micelles in common bile duct ligation (CBDL) induced liver fibrosis, mouse model. We also determined the intrahepatic distribution of fluorescently labeled micelles after MDB5 treatment. Results: Our results show that MDB5 was more potent in inhibiting Hh pathway components and HSC proliferation in vitro. We successfully developed MDB5 loaded micelles with particle size of 40 ± 10 nm and drug loading up to 10% w/w. MDB5 loaded micelles at the dose of 10 mg/kg were well tolerated by mice, without visible sign of toxicity. The serum enzyme activities elevated by CBDL was significantly decreased by MDB5 loaded micelles compared to GDC-0449 loaded micelles. MDB5 loaded micelles further decreased collagen deposition, HSC activation, and Hh activity and its target genes in the liver. MDB5 loaded micelles also prevented liver sinusoidal endothelial capillarization (LSEC) and therefore restored perfusion between blood and liver cells. Conclusions: Our study provides evidence that MDB5 was more potent in inhibiting Hh pathway in HSC-T6 cells and showed better hepatoprotection in CBDL mice compared to GDC-0449.