Multicopy Chromosome Integration and Deletion of Negative Global Regulators Significantly Increased the Heterologous Production of Aborycin in Streptomyces coelicolor.

Aborycin is a type I lasso peptide with a stable interlocked structure, offering a favorable framework for drug development. The aborycin biosynthetic gene cluster gul from marine sponge-associated Streptomyces sp. HNS054 was cloned and integrated into the chromosome of S. coelicolor hosts with different copies. The three-copy gul-integration strain S. coelicolor M1346::3gul showed superior production compared to the one-copy or two-copy gul-integration strains, and the total titer reached approximately 10.4 mg/L, i.e., 2.1 times that of the native strain. Then, five regulatory genes, phoU (SCO4228), wblA (SCO3579), SCO1712, orrA (SCO3008) and gntR (SCO1678), which reportedly have negative effects on secondary metabolism, were further knocked out from the M1346::3gul genome by CRISPR/Cas9 technology. While the ΔSCO1712 mutant showed a significant decrease (4.6 mg/L) and the ΔphoU mutant showed no significant improvement (12.1 mg/L) in aborycin production, the ΔwblA, ΔorrA and ΔgntR mutations significantly improved the aborycin titers to approximately 23.6 mg/L, 56.3 mg/L and 48.2 mg/L, respectively, which were among the highest heterologous yields for lasso peptides in both Escherichia coli systems and Streptomyces systems. Thus, this study provides important clues for future studies on enhancing antibiotic production in Streptomyces systems.

Acute toxicity of Zanthoxylum bungeanum against two stored product insects and synergistic interactions between two major compounds limonene and linalool.

In order to find and develop new botanical pesticides against storage pests, components of the essential oil (EO) from Zanthoxylum bungeanum were identified by GC-MS and their insecticidal activity against the stored product pests were studied. The EO was obtained by steam distillation. Results showed that EO was rich in limonene (23.67), linalool (21.76) and linalyl anthranilate (10.87). In contact assays, linalool exhibited strongest toxicity to red flour beetle adult (LD50 = 17.06 µg/adult) and larvae (LD50 = 16.42 µg/larvae), and linalool was the most active one against the Lasioderma serricorne (LD50 = 15.36 µg/larvae). Then limonene and linalool showed different levels of fumigant activities against the two insect species. Synergism effect existed in the proportion of contact assays against Tribolium castaneum adults, and additive was observed in the proportion of 7:1 against T. castaneum larvae. This work provides important information for the development and utilization of Z. bungeanum and suggests that the EO of Z. bungeanum has the potential to serve as bio-insecticides for controlling pest damage in stored products.

Insecticidal and acetylcholine esterase inhibition activity of Rhododendron thymifolium essential oil and its main constituent against two stored product insects.

In this work, we investigated the bioactivities of the essential oil (EO) extracted from the Rhododendron thymifolium and its principal germacrone against Lasioderma serricorne and Tribolium castaneum. The EO was obtained by steam distillation. Germacrone was obtained by cryogenic crystallization. The bioactivity of EO and germacrone was tested via contact and repellent activity assays. The results showed that EO and germacrone possessed contact and repellent activities against two species of insects. EO exhibited obvious contact activity against the L. serricorn adults, larvae and T. castaneum larvae with LD50 values of 29.15 µg/adult, 42.73 µg/larva, 19.65 µg/larva respectively. Germacrone exhibited excellent contact activity against the L. serricorne adults, larvae and the T. castaneum larvae with LD50 values of 17.18 µg/adult, 20.94 µg/larva, 20.93 µg/larva respectively. And at the highest testing concentrations (78.63 and 15.73 nL/cm2), the repellent activity of EO and germacrone on two target insects was comparable to that of the positive control (DEET) after 30 h exposure. In especially, in the treatment of the 120 h after the repellent activity of EO and germacrone against T.castaneum adults and larvae were still very significant and showed the same level percentage repellency as DEET. Meanwhile, germacrone exhibited inhibition of acetylcholinesterase activity with IC50 values of 3%. The results indicated that the EO of R. thymifolium and germacrone had the potential to be developed as natural insecticides and repellents for the control of T. castaneum and L. serricorne.

Chemical Constituents of Murraya tetramera Huang and Their Repellent Activity against Tribolium castaneum.

Sixteen compounds were isolated from the leaves and stems of Murrayatetramera Huang. Based on the NMR and MS spectral results, the structures were determined. It was confirmed that the isolated compounds included three new compounds (9, 10 and 13) and one new natural product (8), which were identified asmurratetra A (9), murratetra B (10), murratetra C (13) and [2-(7-methoxy-2-oxochromen-8-yl)-3-methylbut-2-enyl]3-methylbut-2-enoate (8), respectively. Meanwhile, the repellent activity against Tribolium castaneum was investigated for 13 of these isolated compounds. The results showed that the tested compounds had various levels of repellent activity against T. castaneum. Among them, compounds 1 (4(15)-eudesmene-1ß,6α-diol), 11 (isoferulic acid) and 16 (2,3-dihydroxypropyl hexadecanoate) showed fair repellent activity against T. castaneum. They might be considered as potential leading compounds for the development of natural repellents.

Chemical Constituents and Insecticidal Activities of Ajania fruticulosa Essential Oil.

The insecticidal activity and chemical constituents of the essential oil from Ajania fruticulosa were investigated. Twelve constituents representing 91.0% of the essential oil were identified, and the main constituents were 1,8-cineole (41.40%), (+)-camphor (32.10%), and myrtenol (8.15%). The essential oil exhibited contact toxicity against Tribolium castaneum and Liposcelis bostrychophila adults with LD50 values of 105.67 µg/adult and 89.85 µg/cm(2) , respectively. The essential oil also showed fumigant toxicity against two species of insect with LC50 values of 11.52 and 0.65 mg/l, respectively. 1,8-Cineole exhibited excellent fumigant toxicity (LC50  = 5.47 mg/l) against T. castaneum. (+)-Camphor showed obvious fumigant toxicity (LC50  = 0.43 mg/l) against L. bostrychophila. Myrtenol showed contact toxicity (LD50  = 29.40 µg/cm(2) ) and fumigant toxicity (LC50  = 0.50 mg/l) against L. bostrychophila. 1,8-Cineole and (+)-camphor showed strong insecticidal activity to some important insects, and they are main constituents of A. fruticulosa essential oil. The two compounds may be related to insecticidal activity of A. fruticulosa essential oil against T. castaneum and L. bostrychophila.

Chemical Composition and Bioactivities of the Essential Oil from Etlingera yunnanensis against Two Stored Product Insects.

The chemical composition of the essential oil of Etlingera yunnanensis rhizomes and its contact and repellent activities against Tribolium castaneum (Herbst) and Liposcelis bostrychophila (Badonnel) were investigated. The essential oil obtained from E. yunnanensis rhizomes with hydrodistillation was performed by gas chromatography-flame ionization detection and gas chromatography-mass spectrometry. The main components of the essential oil were identified to be estragole (65.2%), ß-caryophyllene (6.4%), 1,8-cineole (6.4%), limonene (5.2%), and α-pinene (2.4%). It was found that the essential oil of E. yunnanensis rhizomes possessed contact toxicity against T. castaneum and L. bostrychophila (LD50 = 23.33 µg/adult and LD50 = 47.38 µg/cm², respectively). Estragole, 1,8-cineole, and limonene exhibited stronger contact toxicity (LD50 values of 20.41, 18.86, and 13.40 µg/adult, respectively) than ß-caryophyllene (LD50 = 41.72 µg/adult) against T. castaneum adults. Estragole possessed stronger contact toxicity (LD50 = 30.22 µg/cm²) than ß-caryophyllene, 1,8-cineole, and limonene (LD50 values of 74.11, 321.20, and 239.62 µg/adult, respectively) against L. bostrychophila adults. Repellency of the crude oil was also evaluated. The essential oil and constituents possessed strong repellent activity against T. castaneum adults. The four individual constituents showed weaker repellent activity than the essential oil against L. bostrychophila adults. The results indicated that the essential oil of E. yunnanensis rhizomes and the individual constituents had the potential to be developed as a natural insecticide and repellent for the control of T. castaneum and L. bostrychophila.

[Chemical constituents from leaf of Rhododendron przewalskii].

OBJECTIVE: To study the chemical constituents from the leaf of Rhododendron przewalskii. METHODS: Isolate and purify compounds by chromatographic methods, and identify structures by spectroscopic methods. RESULTS: Six compounds were isolated and identified as hyperin (1), avicularin (2), gossypetin-3-O-ß-D-galactoside (3), quercetin (4), betuloside (5) and ß-sitosterol (6). CONCLUSION: Compounds 1-4 and 6 are isolated from Rhododendron przewalskii for the frist time.

Combination of pharmacophore hypothesis, genetic function approximation model, and molecular docking to identify novel inhibitors of S6K1.

S6K1 has emerged as a potential target for the treatment for obesity, type II diabetes and cancer diseases. Discovery of S6K1 inhibitors has thus attracted much attention in recent years. In this investigation, a hybrid virtual screening method that involves pharmacophore hypothesis, genetic function approximation (GFA) model, and molecular docking technology has been used to discover S6K1 inhibitors especially with novel scaffolds. The common feature pharmacophore hypothesis and GFA regression model of S6K1 inhibitors were first developed and applied in a virtual screen of the Specs database for retrieving S6K1 inhibitors. Then, the molecular docking method was carried out to re-filter these screened compounds. Finally, 60 compounds with promising S6K1 inhibitory activity were carefully selected and have been handed over to the other group to complete the follow-up compound synthesis (or purchase) and activity test.

Toxicity and Synergistic Effect of Elsholtzia Ciliata Essential Oil and Its Main Components Against the Adult and Larval Stages of Tribolium Castaneum.

Investigations have indicated that storage pests pose a great threat to global food security by damaging food crops and other food products derived from plants. Essential oils are proven to have significant effects on a large number of stored grain insects. This study evaluated the contact toxicity and fumigant activity of the essential oil extract from the aerial parts of Elsholtzia ciliata and its two major biochemical components against adults and larvae of the food storage pest beetle Tribolium castaneum. Gas chromatography-mass spectrometry analysis revealed 16 different components derived from the essential oil of E. ciliata, which included carvone (31.63%), limonene (22.05%), and α-caryophyllene (15.47%). Contact toxicity assay showed that the essential oil extract exhibited a microgram-level of killing activity against T. castaneum adults (lethal dose 50 (LD50) = 7.79 µg/adult) and larvae (LD50 = 24.87 µg/larva). Fumigant toxicity assay showed LD50 of 11.61 mg/L air for adults and 8.73 mg/L air for larvae. Carvone and limonene also exhibited various levels of bioactivity. A binary mixture (2:6) of carvone and limonene displayed obvious contact toxicity against T. castaneum adults (LD50 = 10.84 µg/adult) and larvae (LD50 = 30.62 µg/larva). Furthermore, carvone and limonene exhibited synergistic fumigant activity against T. castaneum larvae at a 1:7 ratio. Altogether, our results suggest that E. ciliata essential oil and its two monomers have a potential application value to eliminate T. castaneum.

Chemical constituents isolated from stems of Schisandra chinensis and their antifeedant activity against Tribolium castaneum.

One new sesquiterpene (α-iso-cubebenol acetate, 8), together with 9 known compounds (1-7, 9, 10) were isolated from the stems of Schisandra chinensis (Turcz.) Baill. by repeated silica gel column chromatography. Based on the results of MS, NMR spectra and comparing with literature data, the six dibenzocyclooctadiene lignans were identified as schizandrin A to C (1-3), schizandrin (4), schisantherin A (5) and gomisin J (6), the two sesquiterpenes were identified as α-iso-cubebenol (7) and α-iso-cubebenol acetate (8), while the two triterpenic acids were identified as ganwuweizic acid (9) and kadsuric acid (10). The antifeedant activity of the 10 compounds against Tribolium castaneum adults was tested. Gomisin J (6) exhibited activity at 1500 ppm concentration with 40.3% antifeeding index percentages. As for the dibenzocyclooctene lignans (compounds 1-3, 6), the number of methylenedioxies and the position of hydroxyl groups were the main factors to affect their antifeedant activities.

[Setup and Microbial Community Analysis of ANAMMOX System for Landfill Leachate Treatment Coupling Partial Nitrification-Denitrification Process].

In order to upgrade the current shortcut nitrification and denitrification process for landfill leachate treatment and to stimulate nitrification-denitrification coupled with anaerobic ammonia oxidation (ANAMMOX) at a landfill, an ANAMMOX process was started using an up-flow anaerobic sludge bed (UASB) reactor seeded with nitrification and denitrification sludge. The performances of the reactor were investigated, including the nitrogen loading and nitrogen removal rates. Moreover, Illumina Miseq sequencing was conducted to analyze the microbial community dynamics under long-term operation on a molecular level. The results showed that the ANAMMOX reactor was successfully started in 149 days. The total nitrogen loading rate reached 4000.00 mg·(L·d)-1, and the total nitrogen removal rate reached 3885.76 mg·(L·d)-1 after stable operation. The average ammonium and nitrite removal efficiencies were more than 95%. In 250 days, the Planctomycetes in the reactor experienced rapid growth, and its abundance reached 54.94%. The abundance of Candidatus Kuenenia reached 49.66%. The upgrading process of landfill leachate treatment by coupling ANAMMOX based on short-cut nitrification and denitrification was confirmed to be feasible.

Insecticidal Activities and Chemical Composition of the Essential Oils of Ajania nitida and Ajania nematoloba from China.

In this work, we investigated insecticidal and repellent activities of the essential oils extracted from Ajania nitida and Ajania nematoloba against Tribolium castaneum and Lasioderma serricorne adults. The components of essential oils were analyzed by GC-MS. The main components of A. nitida oil were camphor (20.76%), thujone (18.64%), eucalyptol (13.42%), borneol (8.32%) and those of A. nematoloba oil were ß-pinene (34.72%), eucalyptol (24.97%) and verbenol (20.39%). The results showed that the two essential oils possessed insecticidal and repellent activities against two species of insects. A. nitida oil possessed contact and fumigant toxicity against T. castaneum (LD50 = 30.10 µg/adult and LC50 = 21.07 mg/L air) and L.serricorne (LD50 = 17.51 µg/adult and LC50 = 11.23 mg/L air). A.nematoloba oil showed contact and fumigant toxicity against T. castaneum (LD50 = 102.29µg/adult and LC50 = 69.45 mg/L air) and contact toxicity against L.serricorne (LD50 = 53.43 µg/adult), but no obvious fumigant effect was observed against L.serricorne. Both of essential oils possessed strong repellent activity against T. castaneum and certain repellent activity against L.serricorne. Especially, A. nematoloba oil showed the same level percentage repellency as DEET(the positve control) against T. castaneum. The results indicated that the essential oils of A. nitida and A. nematoloba had the potential to be developed as natural insecticides and repellents for the control of T. castaneum and L.serricorne.

Fumigant and repellent activities of essential oil extracted from Artemisia dubia and its main compounds against two stored product pests.

The major chemical constituents of the essential oil extracted from Artemisia dubia wall. ex Bess. (Family: Asteraceae) were found as terpinolene (19.02%), limonene (17.40%), 2,5-etheno[4.2.2]propella-3,7,9-triene (11.29%), isoelemicin (11.05%) and p-cymene-8-ol (5.93%). Terpinolene and limonene were separated as main components from the essential oil. The essential oil showed fumigant toxicity against Tribolium castaneum and Liposcelis bostrychophila with LC50 values of 49.54 and 0.74 mg/L, respectively. The essential oil and isolated compounds of A. dubia showed repellency activities against both insects. Terpinolene and limonene showed the fumigant toxicity against T. castaneum. Terpinolene showed obvious fumigant toxicity against L. bostrychophila. The results indicated that the essential oil of A. dubia had potential to be developed into natural insecticides for controlling stored product pests.

[Alkaloid constituents of Corydalis adunca].

OBJECTIVE: To study the alkaloid component of Corydalis adunca Maxim. METHODS: The constituents were isolated by chromatographic methods, their structures were elucidated by spectroscopic evidences. RESULTS: Three compounds were purified and their structures were identified, they were identified as dorydaline (I), dehydrocorydaline (II), dihydrosanguinarine (III). CONCLUSION: Compounds I , II are isolated from this plant for the first time.

Bioactivities and Chemical Constituents of Essential Oil Extracted from Artemisia anethoides Against Two Stored Product Insects.

The chemical constituents of the essential oil extracted from Artemisia anethoides and the bioactivities of essential oil against Tribolium castaneum and Lasioderma serricorne were investigated. The main components of the essential oil were 1,8-cineole (36.54%), 2-isopropyl-5-methyl-3-cyclohexen-1-one (10.40%), terpinen-4-ol (8.58%), 2-isopropyltoluene (6.20) and pinocarveol (5.08%). The essential oil of A. anethoides possessed contact and fumigant toxicities against T. castaneum adults (LD50 = 28.80 µg/adult and LC50 = 13.05 mg/L air, respectively) and against L. serricorne (LD50 = 24.03 µg/adult and LD50 = 8.04 mg/L air, respectively). The crude oil showed repellent activity against T. castaneum and L. serricorne. Especially, the percentage repellency of essential oil was same level with DEET (positive control) against T. castaneum. The results indicated that the essential oil of A. anethoides had the potential to be developed as insecticide and repellent for control of T. castaneum and L. serricorne.

Development of novel in silico model for developmental toxicity assessment by using naïve Bayes classifier method.

Toxicological testing associated with developmental toxicity endpoints are very expensive, time consuming and labor intensive. Thus, developing alternative approaches for developmental toxicity testing is an important and urgent task in the drug development filed. In this investigation, the naïve Bayes classifier was applied to develop a novel prediction model for developmental toxicity. The established prediction model was evaluated by the internal 5-fold cross validation and external test set. The overall prediction results for the internal 5-fold cross validation of the training set and external test set were 96.6% and 82.8%, respectively. In addition, four simple descriptors and some representative substructures of developmental toxicants were identified. Thus, we hope the established in silico prediction model could be used as alternative method for toxicological assessment. And these obtained molecular information could afford a deeper understanding on the developmental toxicants, and provide guidance for medicinal chemists working in drug discovery and lead optimization.

Novel naïve Bayes classification models for predicting the chemical Ames mutagenicity.

Prediction of drug candidates for mutagenicity is a regulatory requirement since mutagenic compounds could pose a toxic risk to humans. The aim of this investigation was to develop a novel prediction model of mutagenicity by using a naïve Bayes classifier. The established model was validated by the internal 5-fold cross validation and external test sets. For comparison, the recursive partitioning classifier prediction model was also established and other various reported prediction models of mutagenicity were collected. Among these methods, the prediction performance of naïve Bayes classifier established here displayed very well and stable, which yielded average overall prediction accuracies for the internal 5-fold cross validation of the training set and external test set I set were 89.1±0.4% and 77.3±1.5%, respectively. The concordance of the external test set II with 446 marketed drugs was 90.9±0.3%. In addition, four simple molecular descriptors (e.g., Apol, No. of H donors, Num-Rings and Wiener) related to mutagenicity and five representative substructures of mutagens (e.g., aromatic nitro, hydroxyl amine, nitroso, aromatic amine and N-methyl-N-methylenemethanaminum) produced by ECFP_14 fingerprints were identified. We hope the established naïve Bayes prediction model can be applied to risk assessment processes; and the obtained important information of mutagenic chemicals can guide the design of chemical libraries for hit and lead optimization.

Europium (III) chelate microparticle-based lateral flow immunoassay strips for rapid and quantitative detection of antibody to hepatitis B core antigen.

Quantitative hepatitis B core antigen (anti-HBc) measurements could play an important role in evaluating therapeutic outcomes and optimizing the antiviral therapy of chronic hepatitis B infection. In this study, we have developed a simple and rapid fluorescence point-of-care test based on a lateral flow immunoassay (LFIA) method integrated with Eu (III) chelate microparticles to quantitatively determine anti-HBc concentrations in serum. This assay is based on a direct competitive immunoassay performed on lateral flow test strips with an assay time of 15 min. The Eu (III) chelate microparticle-based LFIA assay could quantitatively detect anti-HBc levels with a limit of detection of 0.31 IU mL-1, and exhibited a wide linear range (0.63-640 IU mL-1). The intra- and inter-assay coefficients of variation for anti-HBc were both less than 10% and a satisfactory dilution test and accuracy were demonstrated. There were no statistically significant differences in sensitivity or specificity in serum samples between the Eu (III) chelate microparticle-based LFIA strips and the Abbott Architect kit. A simple, rapid and effective quantitative detection of anti-HBc was possible using the Eu (III) chelate microparticle-based LFIA strips. The strips will provide diagnostic value for clinical application.

Repellence of the main components from the essential oil of Glycosmis lucida Wall. ex Huang against two stored product insects.

A screening of Chinese medicinal herbs and wild plants for agrochemicals was carried out; the essential oil of Glycosmis lucida leaves was found to possess significant repellent activity against Tribolium castaneum and Liposcelis bostrychophila. It was found that the main components included elixene (19.81%), spathulenol (10.68%), anethole (12.05%), verbenone (10.32%) followed by ß-caryophyllene (6.87%). The essential oil, anethole and verbenone were strongly repellent against T. castaneum (96, 86 and 94%, respectively, at 15.73 nL cm-2) and L. bostrychophila (100, 68 and 72%, respectively, at 31.58 nL cm-2) after a 2h treatment. The results indicate that anethole and verbenone had the potential to be developed as natural repellents for control of stored product insects.