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Background: Abamectin (ABA) is considered a powerful insecticidal and anthelmintic agent. It is an intracellular product of Streptomyces avermitilis; is synthesized through complicated pathways and can then be extracted from mycelial by methanol extraction. ABA serves as a biological control substance against the root-knot nematode Meloidogyne incognita. This investigation is intended to reach a new strain of S. avermitilis capable of producing ABA effectively., Results: Among the sixty actinobacterial isolates, Streptomyces St.53 isolate was chosen for its superior nematicidal effectiveness. The mycelial-methanol extract of isolate St.53 exhibited a maximum in vitro mortality of 100% in one day. In the greenhouse experiment, the mycelial-methanol extract demonstrated, for the second-stage juveniles (J 2 s), 75.69% nematode reduction and 0.84 reproduction rate (Rr) while for the second-stage juveniles (J 2 s), the culture suspension demonstrated 75.38% nematode reduction and 0.80 reproduction rate (Rr). Molecular identification for St.53 was performed using 16 S rRNA gene analysis and recorded in NCBI Genbank as S. avermitilis MICNEMA2022 with accession number (OP108264.1). LC-MS was utilized to detect and identify abamectin in extracts while HPLC analysis was carried out for quantitative determination. Both abamectin B1a and abamectin B1b were produced and detected at retention times of 4.572 and 3.890 min respectively., Conclusion: Streptomyces avermitilis MICNEMA2022 proved to be an effective source for producing abamectin as a biorational agent for integrated nematode management., (© 2024. The Author(s).)

The glutathione S -transferases (GSTs) are important detoxifying enzymes in insects. Our previous studies found that the susceptibility of Chilo suppressalis to abamectin was significantly increased when the CsGST activity was inhibited by glutathione (GSH) depletory. In this study, the potential detoxification mechanisms of CsGSTs to abamectin were explored. Six CsGSTs of C. suppressalis were expressed in vitro . Enzymatic kinetic parameters including K m and V max of recombinant CsGSTs were determined, and results showed that all of the six CsGSTs were catalytically active and displaying glutathione transferase activity. Insecticide inhibitions revealed that a low concentration of abamectin could effectively inhibit the activities of CsGSTs including CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1. However, the in vitro metabolism assay found that the six CsGSTs could not metabolize abamectin directly. Additionally, the glutathione transferase activity of CsGSTs in C. suppressalis was significantly increased post-treatment with abamectin. Comprehensive analysis of the results in present and our previous studies demonstrated that CsGSTs play an important role in detoxification of abamectin by catalyzing the conjugation of GSH to abamectin in C. suppressalis , and the high binding affinities of CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1 with abamectin might also suggest the involvement of CsGSTs in detoxification of abamectin via the noncatalytic passive binding and sequestration instead of direct metabolism. These studies are helpful to better understand the detoxification mechanisms of GSTs in insects.

Doramectin, an essential animal anthelmintic, is synthesized through the fermentation process of Streptomyces avermitilis. This study delves into the transcriptomic profiles of two strains, namely the doramectin-producing wild-type S. avermitilis N72 and its highly doramectin-producing mutant counterpart, S. avermitilis XY-62. Comparative analysis revealed 860 up-regulated genes and 762 down-regulated genes in the mutant strain, notably impacting the expression of key genes pivotal in doramectin biosynthesis, including aveA1, aveA2, aveA3, aveA4, aveE, and aveBI. These findings shed light on the molecular mechanisms underpinning the heightened doramectin production in S. avermitilis XY-62, presenting promising avenues for optimizing doramectin production processes., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Pine wood nematode (PWN) disease is a globally devastating forest disease caused by infestation with PWN, Bursaphelenchus xylophilus , which mainly occurs through the vector insect Japanese pine sawyer (JPS), Monochamus alternatus . PWN disease is notoriously difficult to manage effectively and is known as the "cancer of pine trees." In this study, dual enzyme-responsive nanopesticides (AVM@EC@Pectin) were prepared using nanocoating avermectin (AVM) after modification with natural polymers. The proposed treatment can respond to the cell wall-degrading enzymes secreted by PWNs and vector insects during pine tree infestation to intelligently release pesticides to cut off the transmission and infestation pathways and realize the integrated control of PWN disease. The LC 50 value of AVM@EC@Pectin was 11.19 mg/L for PWN and 26.31 mg/L for JPS. The insecticidal activity of AVM@EC@Pectin was higher than that of the commercial emulsifiable concentrate (AVM-EC), and the photostability, adhesion, and target penetration were improved. The half-life ( t 1/2 ) of AVM@EC@Pectin was 133.7 min, which is approximately twice that of AVM-EC (68.2 min). Sprayed and injected applications showed that nanopesticides had superior bidirectional transportation, with five-times higher AVM contents detected in the roots relative to those of AVM-EC when sprayed at the top. The safety experiment showed that the proposed treatment had lower toxicity and higher safety for nontarget organisms in the application environment and human cells. This study presents a green, safe, and effective strategy for the integrated management of PWN disease.

Guvermectin, as a novel nucleoside-like biopesticide, could increase the rice yield excellently, but the potential environmental behaviors remain unclear, which pose potential health risks. Therefore, the uptake and biotransformation of guvermectin in three types of crops (rice, lettuce, and carrot) were first evaluated with a hydroponic system. Guvermectin could be rapidly absorbed and reached equilibrium in roots (12-36 h) and shoots (24-60 h) in three plants, and guvermectin was also vulnerable to dissipation in roots ( t 1/2 1.02-3.65 h) and shoots ( t 1/2 9.30-17.91 h). In addition, 8 phase I and 2 phase II metabolites, transformed from guvermectin degradation in vivo and in vitro exposure, were identified, and one was confirmed as psicofuranine, which had antibacterial and antitumor properties; other metabolites were nucleoside-like chemicals. Molecular simulation and quantitative polymerase chain reaction further demonstrated that guvermectin was metabolized by the catabolism pathway of an endogenous nucleotide. Guvermectin had similar metabolites in three plants, but the biotransformation ability had a strong species dependence. In addition, all the metabolites exhibit neglectable toxicities (bioconcentration factor <2000 L/kg b.w., LC 50,rat > 5000 mg/kg b.w.) by prediction. The study provided valuable evidence for the application of guvermectin and a better understanding of the biological behavior of nucleoside-like pesticides.

Avermectins (AVEs), a family of macrocyclic polyketides produced by Streptomyces avermitilis, have eight components, among which B1a is noted for its strong insecticidal activity. Biosynthesis of AVE "a" components requires 2-methylbutyryl-CoA (MBCoA) as starter unit, and malonyl-CoA (MalCoA) and methylmalonyl-CoA (MMCoA) as extender units. We describe here a novel strategy for increasing B1a production by enhancing acyl-CoA precursor supply. First, we engineered meilingmycin (MEI) polyketide synthase (PKS) for increasing MBCoA precursor supply. The loading module (using acetyl-CoA as substrate), extension module 7 (using MMCoA as substrate) and TE domain of MEI PKS were assembled to produce 2-methylbutyrate, providing the starter unit for B1a production. Heterologous expression of the newly designed PKS (termed Mei-PKS) in S. avermitilis wild-type (WT) strain increased MBCoA level, leading to B1a titer 262.2 μg/mL - 4.36-fold higher than WT value (48.9 μg/mL). Next, we separately inhibited three key nodes in essential pathways using CRISPRi to increase MalCoA and MMCoA levels in WT. The resulting strains all showed increased B1a titer. Combined inhibition of these key nodes in Mei-PKS expression strain increased B1a titer to 341.9 μg/mL. Overexpression of fatty acid β-oxidation pathway genes in the strain further increased B1a titer to 452.8 μg/mL - 8.25-fold higher than WT value. Finally, we applied our precursor supply strategies to high-yield industrial strain A229. The strategies, in combination, led to B1a titer 8836.4 μg/mL - 37.8% higher than parental A229 value. These findings provide an effective combination strategy for increasing AVE B1a production in WT and industrial S. avermitilis strains, and our precursor supply strategies can be readily adapted for overproduction of other polyketides., (© 2024 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd.)

Background: The macrolide antibiotic avermectin, a natural product derived from Streptomyces avermitilis, finds extensive applications in agriculture, animal husbandry and medicine. The mtrA (sav&#95;5063) gene functions as a transcriptional regulator belonging to the OmpR family. As a pleiotropic regulator, mtrA not only influences the growth, development, and morphological differentiation of strains but also modulates genes associated with primary metabolism. However, the regulatory role of MtrA in avermectin biosynthesis remains to be elucidated., Results: In this study, we demonstrated that MtrA, a novel OmpR-family transcriptional regulator in S. avermitilis, exerts global regulator effects by negatively regulating avermectin biosynthesis and cell growth while positively controlling morphological differentiation. The deletion of the mtrA gene resulted in an increase in avermectin production, accompanied by a reduction in biomass and a delay in the formation of aerial hyphae and spores. The Electrophoretic Mobility Shift Assay (EMSA) revealed that MtrA exhibited binding affinity towards the upstream region of aveR, the intergenic region between aveA1 and aveA2 genes, as well as the upstream region of aveBVIII in vitro. These findings suggest that MtrA exerts a negative regulatory effect on avermectin biosynthesis by modulating the expression of avermectin biosynthesis cluster genes. Transcriptome sequencing and fluorescence quantitative PCR analysis showed that mtrA deletion increased the transcript levels of the cluster genes aveR, aveA1, aveA2, aveC, aveE, aveA4 and orf-1, which explains the observed increase in avermectin production in the knockout strain. Furthermore, our findings demonstrate that MtrA positively regulates the cell division and differentiation genes bldM and ssgC, while exerting a negative regulatory effect on bldD, thereby modulating the primary metabolic processes associated with cell division, differentiation and growth in S. avermitilis, consequently impacting avermectin biosynthesis., Conclusions: In this study, we investigated the negative regulatory effect of the global regulator MtrA on avermectin biosynthesis and its effects on morphological differentiation and cell growth, and elucidated its transcriptional regulatory mechanism. Our findings indicate that MtrA plays crucial roles not only in the biosynthesis of avermectin but also in coordinating intricate physiological processes in S. avermitilis. These findings provide insights into the synthesis of avermectin and shed light on the primary and secondary metabolism of S. avermitilis mediated by OmpR-family regulators., (© 2024. The Author(s).)

Parasitic nematodes pose a significant threat to human and animal health, as well as cause economic losses in the agricultural sector. The use of anthelmintic drugs, such as Ivermectin (IVM), to control these parasites has led to widespread drug resistance. Identifying genetic markers of resistance in parasitic nematodes can be challenging, but the free-living nematode Caenorhabditis elegans provides a suitable model. In this study, we aimed to analyze the transcriptomes of adult C. elegans worms of the N2 strain exposed to the anthelmintic drug Ivermectin (IVM), and compare them to those of the resistant strain DA1316 and the recently identified Abamectin Quantitative Trait Loci (QTL) on chromosome V. We exposed pools of 300 adult N2 worms to IVM (10-7 and 10-8 M) for 4 hours at 20°C, extracted total RNA and sequenced it on the Illumina NovaSeq6000 platform. Differentially expressed genes (DEGs) were determined using an in-house pipeline. The DEGs were compared to genes from a previous microarray study on IVM-resistant C. elegans and Abamectin-QTL. Our results revealed 615 DEGs (183 up-regulated and 432 down-regulated genes) from diverse gene families in the N2 C. elegans strain. Of these DEGs, 31 overlapped with genes from IVM-exposed adult worms of the DA1316 strain. We identified 19 genes, including the folate transporter (folt-2) and the transmembrane transporter (T22F3.11), which exhibited an opposite expression in N2 and the DA1316 strain and were deemed potential candidates. Additionally, we compiled a list of potential candidates for further research including T-type calcium channel (cca-1), potassium chloride cotransporter (kcc-2), as well as other genes such as glutamate-gated channel (glc-1) that mapped to the Abamectin-QTL., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Dube et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The α7 nicotinic acetylcholine receptor (α7nAChR) mediates signaling in the central nervous system and cholinergic anti-inflammatory pathways. Ivermectin is a positive allosteric modulator of a full-length α7nAChR and an agonist of the α7nAChR construct containing transmembrane (TMD) and intracellular (ICD) domains, but structural insights of the binding have not previously been determined. Here, combining nuclear magnetic resonance as a primary experimental tool with Rosetta comparative modeling and molecular dynamics simulations, we have revealed details of ivermectin binding to the α7nAChR TMD + ICD and corresponding structural changes in an ivermectin-induced desensitized state. Ivermectin binding was stabilized predominantly by hydrophobic interactions from interfacial residues between adjacent subunits near the extracellular end of the TMD, where the inter-subunit gap was substantially expanded in comparison to the apo structure. The ion-permeation pathway showed a profile distinctly different from the resting-state profile but similar to profiles of desensitized α7nAChR. The ICD also exhibited structural changes, including reorientation of the MX and h3 helices relative to the channel axis. The resulting structures of the α7nAChR TMD + ICD in complex with ivermectin provide opportunities for discovering new modulators of therapeutic potential and exploring the structural basis of cytoplasmic signaling under different α7nAChR functional states.

Glioblastoma (GBM) is one of the most widespread and lethal types of cancer. However, there are currently no drugs or therapeutic strategies that can completely cure GBM. Doramectin (DRM) has a broad range of activities against endoparasites and ectoparasites, and is extensively used in livestock. In the present study, the effect of DRM on the induction of autophagy in U87 and C6 GBM and glioma cell lines, as well as the mechanism of autophagy, were examined. First, transmission electron microscopy, plasmid transfection and western blot analysis demonstrated that DRM could induce autophagy in U87 and C6 cells in vitro . Next, MTT and colony formation assays revealed that DRM‑induced autophagy prevented U87 and C6 cell viability and colony formation ratio. In addition, DRM‑induced autophagy promoted U87 and C6 cell apoptosis, as indicated by DAPI analysis and flow cytometry. Furthermore, transcriptome analysis demonstrated that DRM modulated a number of genes and pathways involved in autophagy. In a nude mouse xenograft model, immunohistochemical staining and the TUNEL assay demonstrated that the effect of DRM on the tumor was consistent with that in vivo . These data indicated that DRM induced autophagy mainly by blocking the PI3K/AKT/mTOR signaling pathway in GBM cells. DRM‑induced autophagy promoted the inhibition of GBM cell proliferation and apoptosis in vitro and in vivo . The present study suggested that DRM may be an effective drug for the treatment of GBM.

The identification of chemical compounds able to bind specific sites of the human/viral proteins involved in the SARS-CoV-2 infection cycle is a prerequisite to design effective antiviral drugs. Here we conduct a molecular dynamics study with the aim to assess the interactions of ivermectin, an antiparasitic drug with broad-spectrum antiviral activity, with the human Angiotensin-Converting Enzyme 2 (ACE2), the viral 3CL pro and PL pro proteases, and the viral SARS Unique Domain (SUD). The drug/target interactions have been characterized in silico by describing the nature of the non-covalent interactions found and by measuring the extent of their time duration along the MD simulation. Results reveal that the ACE2 protein and the ACE2/RBD aggregates form the most persistent interactions with ivermectin, while the binding with the remaining viral proteins is more limited and unspecific.

The heat shock response (HSR) is a universal cellular response that promotes survival following temperature increase. In filamentous Streptomyces , which accounts for ∼70% of commercial antibiotic production, HSR is regulated by transcriptional repressors; in particular, the widespread MerR-family regulator HspR has been identified as a key repressor. However, functions of HspR in other biological processes are unknown. The present study demonstrates that HspR pleiotropically controls avermectin production, morphological development, and heat shock and H 2 O 2 stress responses in the industrially important species Streptomyces avermitilis. HspR directly activated ave structural genes ( aveA1 and aveA2 ) and H 2 O 2 stress-related genes ( katA1 , catR , katA3 , oxyR , ahpC , and ahpD ), whereas it directly repressed heat shock genes (HSGs) (the dnaK1-grpE1-dnaJ1-hspR operon, clpB1p , clpB2p , and lonAp ) and developmental genes ( wblB , ssgY , and ftsH ). HspR interacted with PhoP (response regulator of the widespread PhoPR two-component system) at dnaK1p to corepress the important dnaK1-grpE1-dnaJ1-hspR operon. PhoP exclusively repressed target HSGs ( htpG , hsp18&#95;1 , and hsp18&#95;2 ) different from those of HspR ( clpB1p , clpB2p , and lonAp ). A consensus HspR-binding site, 5'-TTGANBBNNHNNNDSTSHN-3', was identified within HspR target promoter regions, allowing prediction of the HspR regulon involved in broad cellular functions. Taken together, our findings demonstrate a key role of HspR in the coordination of a variety of important biological processes in Streptomyces species. IMPORTANCE Our findings are significant to clarify the molecular mechanisms underlying HspR function in Streptomyces antibiotic production, development, and H 2 O 2 stress responses through direct control of its target genes associated with these biological processes. HspR homologs described to date function as transcriptional repressors but not as activators. The results of the present study demonstrate that HspR acts as a dual repressor/activator. PhoP cross talks with HspR at dnaK1p to coregulate the heat shock response (HSR), but it also has its own specific target heat shock genes (HSGs). The novel role of PhoP in the HSR further demonstrates the importance of this regulator in Streptomyces . Overexpression of hspR strongly enhanced avermectin production in Streptomyces avermitilis wild-type and industrial strains. These findings provide new insights into the regulatory roles and mechanisms of HspR and PhoP and facilitate methods for antibiotic overproduction in Streptomyces species.

The review presents metabolic properties of Ivermectin (IVM) as substrate and inhibitor of human P450 (P450, CYP) enzymes and drug transporters. IVM is metabolized, both in vivo and in vitro, by C-hydroxylation and O-demethylation reactions catalyzed by P450 3A4 as the major enzyme, with a contribution of P450 3A5 and 2C9. In samples from both in vitro and in vivo metabolism, a number of metabolites were detected and as major identified metabolites were 3″-O-demethylated, C4-methyl hydroxylated, C25 isobutyl-/isopropyl-hydroxylated, and products of oxidation reactions. Ivermectin inhibited P450 2C9, 2C19, 2D6, and CYP3A4 with IC 50 values ranging from 5.3 μM to no inhibition suggesting that it is no or weak inhibitor of the enzymes. It is suggested that P-gp (MDR1) transporter participate in IVM efflux at low drug concentration with a slow transport rate. At the higher, micromolar concentration range, which saturates MDR1 (P-gp), MRP1, and to a lesser extent, MRP2 and MRP3 participate in IVM transport across physiological barriers. IVM exerts a potent inhibition of P-gp (ABCB1), MRP1 (ABCC1), MRP2 (ABCC2), and BCRP1 (ABCG2), and medium to weak inhibition of OATP1B1 (SLC21A6) and OATP1B3 (SLCOB3) transport activity. The metabolic and transport properties of IVM indicate that when IVM is co-administered with other drugs/chemicals that are potent inhibitors/inducers P4503A4 enzyme and of MDR1 (P-gp), BCRP or MRP transporters, or when polymorphisms of the drug transporters and P450 3A4 exist, drug-drug or drug-toxic chemical interactions might result in suboptimal response to the therapy or to toxic effects.

This study aims to evaluate the influence of the phase behavior of microemulsions in the transdermal administration ("spot-on") of ivermectin, an antiparasitic drug widely used in the treatment of endoparasites and ectoparasites in dogs. In this regard, pseudoternary phase diagrams composed of water (aqueous phase), isopropyl myristate (oil phase), tween 80 (surfactant) and labrasol (cosurfactant) were obtained in a different surfactant: cosurfactant (S:CS) ratios. S:CS in 1:3 ratio presented a larger region of microemulsion formation and three microemulsions were selected from it and characterized. Subsequently, in vitro permeation and retention studies were conducted using canine skin as membrane. SAXS, rheology and conductivity data were employed to confirm the phase behavior of the microemulsions (w/o, bicontinuous or o/w). The cutaneous permeation and retention tests showed that the w/o microemulsion, followed by bicontinuous microemulsion, resulted in a higher amount of drug permeated through canine skin, suggesting better transdermal permeation. On the other hand, o/w microemulsion resulted in a higher amount of drug accumulated into the skin, suggesting better topical activity. Thus, it can be concluded that phase behavior of microemulsions influenced the drug permeation in the canine skin differently from other animal models. Microemulsions, especially w/o and bicontinuous, can be promising vehicles regarding the transdermal delivery of ivermectin., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A terrestrial test system to investigate the biomagnification potential and tissue-specific distribution of ivermectin, a widely used parasiticide, in the non-target dung beetle Thorectes lusitanicus (Jekel) was developed and validated. Biomagnification kinetics of ivermectin in T. lusitanicus was investigated by following uptake, elimination, and distribution of the compound in dung beetles feeding on contaminated faeces. Results showed that ivermectin was biomagnified in adults of T. lusitanicus when exposed to non-lethal doses via food uptake. Ivermectin was quickly transferred from the gut to the haemolymph, generating a biomagnification factor (BMF k ) three times higher in the haemolymph than in the gut after an uptake period of 12 days. The fat body appeared to exert a major role on the biomagnification of ivermectin in the insect body, showing a BMF k 1.6 times higher than in the haemolymph. The results of this study highlight that the biomagnification of ivermectin should be investigated from a global dung-based food web perspective and that the use of these antiparasitic substances should be monitored and controlled on a precautionary basis. Thus, we suggest that an additional effort be made in the development of standardised regulatory recommendations to guide biomagnification studies in terrestrial organisms, but also that it is necessary to adapt existing methods to assess the effects of such veterinary medical products.

Ivermectin is a powerful endectocide, which reduces the incidence of vector-borne diseases. Besides its strong insecticidal effect on mosquito vectors of the disease, ivermectin inhibits Plasmodium falciparum sporogonic and blood stage development and impairs Plasmodium berghei development inside hepatocytes, both in vitro and in vivo. Herein, we present the first report on structural modification of ivermectin to produce dual-action molecular hybrids with good structure-dependent in vitro activity against both the hepatic and erythrocytic stages of P. berghei and P. falciparum infection, suggesting inclusion of ivermectin antimalarial hybrids in malaria control strategies. The most active hybrid displayed over threefold and 10-fold higher in vitro activity than ivermectin against hepatic and blood stage infections, respectively. Although an overwhelming insecticidal effect against Anopheles stephensi mosquitoes in laboratory conditions was not noticed, in silico docking analysis supports allosteric binding to glutamate-gated chloride channels similar to ivermectin.

Novel enzyme cum pH dual-responsive microcapsule were prepared with an aim to enhance the utilization of pesticides and reduce environmental pollution. The synthetic procedure involved the introduction of urea bonds by forming a covalent bond between isocyanate-functionalized polydopamine (PDA) microcapsules and polyethyleneimine (PEI). The prepared AVM@PDA-IPTS-PEI microcapsules could effectively protect avermectin (AVM) from ultraviolet radiation, and thus decelerate the decomposition rate; the cumulative release showed positive correlations with pH and the urease activity. The AVM release rate was highest at pH 10. At pH 7, the release rate was enhanced after addition of urease, showing improved urease-responsive property under neutral or weak base condition. Especially at pH 7 in presence of urease, the release behavior was in agreement with the Logistic model and able to describe the S-shaped/sigmoidal release profiles, whereas Korsmeyer-Peppas model was better suited in the absence of urease, as the value of K 1 was smaller than 0.45, suggesting that the behavior was controlled by Fick's diffusion. AVM@PDA-IPTS-PEI displayed better adhesion property than AVM, and the microcapsule ameliorated foliage wettability. AVM@PDA-IPTS-PEI exhibited the insecticidal property similarly with AVM, which can be used for reducing the applied quantity of pesticides and improving their efficacy., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The experiment developed the ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method for testing emamectin benzoate, and studied the metabolism of emamectin benzoate in rice plants and rice-growing environments via application of this testing method. The dissipation curve of emamectin benzoate standard substance was good at 0.5-200 μg L -1 , and its correlation coefficient was greater than 0.99. In the concentration range of 0.1-50 μg kg -1 , the average recovery rate of plants, soil, and field water was 82 %-102 %, and relative standard deviation (RSD) was between 0.3 % and 15.9 %. Half-lives in rice plants and soil were 0.8-2.8 days and 1.9-3.8 days, respectively, and emamectin benzoate was not detected in rice or rice hull. The experiment showed that emamectin benzoate is harmless to human health at the concentration recommended by the manufacturer., Competing Interests: The authors declare no conflict of interest.

Avermectins and moxidectin are antiparasitics widely used as active pharmaceutical ingredients in veterinary medicine, as well as in pesticide formulations for pest control in agriculture. Although the use of these compounds provides benefits to agribusiness, they can impact the environment, since a large part of these substances may reach the soil and water from the excreta of treated animals and following direct applications to crops. The present work had the objective of evaluating the dissipation behaviors of abamectin, doramectin, eprinomectin, ivermectin, and moxidectin in four native Brazilian soils of different textural classes (clay, sandy-clay, sandy, and sandy-clay-loam), following OECD Guideline 307. The studies were conducted in a climate chamber at 22 °C, 71% relative humidity, and protected from light. The dissipation studies were carried out with all drugs together, since no difference was verified when studies were done with each drug separately. The concentrations of the drugs in the soils were determined using an ultra-high performance liquid chromatograph coupled to a fluorescence detector or a tandem mass spectrometer. The dissipation half-life (DT 50 ) values ranged from 9 to 16 days and the calculated GUS index values were in the range from -1.10 to 0.08, indicating low mobility of the drugs in the soils evaluated and low tendency for leaching. In addition, a field study was carried out to evaluate the dissipation of abamectin after application of a foliar pesticide in an orange crop. A DT 50 of 9 days was determined, which was similar to that obtained under controlled conditions in the climate chamber (12 days), indicating that biotransformation was the primary process influencing the overall dissipation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Veterinary drugs enter the environment in many ways and may affect non-target organisms, including plants. The present project was focused on the biotransformation of ivermectin (IVM), one of the mostly used anthelmintics, in the model plant Arabidopsis thaliana. Our results certified the ability of plants to uptake IVM by roots and translocate it to the aboveground parts. Using UHPLC-MS/MS, six metabolites in roots and only the parent drug in rosettes were found after 24- and 72-h incubation of A. thaliana with IVM. The metabolites were formed only via hydroxylation and demethylation, with no IVM conjugates detected. Although IVM did not induce changes in the activity of antioxidant enzymes in A. thaliana rosettes, the expression of genes was significantly affected. Surprisingly, a higher number of transcripts, 300 and 438, respectively, was dysregulated in the rosettes than in roots. The significantly affected genes play role in response to salt, osmotic and water deprivation stress, in response to pathogens and in ion homeostasis. We hypothesize that the above described changes in gene transcription in A. thaliana resulted from disrupted ionic homeostasis caused by certain ionophore properties of IVM. Our results underlined the negative impact of IVM presence in the environment., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Streptomyces avermitilis is well known as the producer of anthelmintic agent avermectins, which are widely used in agriculture, veterinary medicine, and human medicine. aveI encodes a TetR-family regulator, which is the homolog of AtrA. It was reported that deletion of aveI caused enhanced avermectin production. In this study, we investigated the regulatory function of the AveI in S. avermitilis. By binding to the 15-nt palindromic sequence in the promoter regions, AveI directly regulates at least 35 genes. AveI represses avermectin production by directly regulating the transcription of the cluster-situated regulator gene aveR and structural genes aveA1, aveA3, and aveD. AveI represses oligomycin production by repressing the CSR gene olmRII and structural genes olmC. AveI activates melanin biosynthesis by activating the expression of melC1C2 operon. AveI activates morphological differentiation by activating the expression of ssgR and ssgD genes, repressing the expression of wblI gene. Besides, AveI regulates many genes involved in primary metabolism, including substrates transport, the metabolism of amino acids, lipids, and carbohydrates. Therefore, AveI functions as a global regulator in S. avermitilis, controls not only secondary metabolism and morphological differentiation, but also primary metabolism.

A series of new doramectin derivatives containing carbamate, ester and sulfonate were synthesized, and their structures were characterized by 1 H and 13 C nuclear magnetic resonance (NMR) and high-resolution mass spectrum (HRMS). Their insecticidal activities against oriental armyworm, diamondback moth, and corn borer were evaluated and compared with the parent doramectin and commercial avermectins, metolcarb, fenpropathrin. Among all compounds, three compounds (3a, 3g and 3h) showed excellent insecticidal effect. In particular, compound 3g containing cyclopropyl carbamate against oriental armyworm, diamondback moth, and corn borer, exhibited the most promising insecticidal activity with the final mortality rate of 66.67%, 36.67%, 40.00% at the concentration of 12.5 mg/L, respectively. The LC 50 values of 3g were 5.8859, 22.3214, and 22.0205 mg/L, showing 6.74, 2.23, 2.21-fold higher potency than parent doramectin (LC 50 values of 39.6907, 49.7736, and 48.6129 mg/L) and 6.83, 1.93, 3.36-fold higher potency than commercial avermectins (LC 50 values of 40.2489, 42.9922, and 73.9508 mg/L). Additionally, molecular docking simulations revealed that 3g displayed stronger hydrogen-bonding action in binding with the GABA receptor than parent doramectin, which were crucial for keeping high insecticidal activity. The present work demonstrated that these compounds containing alkyl carbamate group could be considered as potential candidates for the development of novel pesticides in the future., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Veterinary medicinal products (VMPs) are widely used within the fish farming industry to control sea lice infestations. There is concern that wild and farmed mussels in the vicinity to these fish farms may be exposed and subsequently bioaccumulate these chemicals, which could pose a threat to human health. To understand the fate of these chemicals in the environment, controlled laboratory exposures were performed to establish the uptake and depuration of selected VMPs in the blue mussel (Mytilus edulis). The VMPs included teflubenzuron, emamectin benzoate and deltamethrin. The effects of salinity on the bioaccumulation of teflubenzuron were also investigated to see whether mussels in brackish waters exhibit different bioaccumulation dynamics. Salinity had no significant effect on the uptake or depuration curves for teflubenzuron down to 15‰. The uptake rate constants (k 1 ) for teflubenzuron, emamectin benzoate and deltamethrin in mussels were 192, 4.82 and 2003, with kinetic bioconcentration factors (BCFs) of 1304, 49 and 2516. Depuration rate constants (k 2 ) were also found to differ between the three VMPs at 0.147, 0.048 and 0.796 for teflubenzuron, emamectin benzoate and deltamethrin, with calculated elimination half-lives (t 1/2 )of 4.7, 14 and 0.87 days. The longer elimination half-lives for teflubenzuron and emamectin benzoate, suggest that these chemicals accumulate in blue mussels and therefore have the potential to bioaccumulate in wild and farmed mussel populations in the environment., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Resistance to the anthelmintic macrocyclic lactone ivermectin (IVM) has a great impact on the control of parasitic nematodes. The mechanisms by which nematodes adapt to IVM remain to be deciphered. We have identified NHR-8, a nuclear hormone receptor involved in the xenobiotic response in Caenorhabditis elegans, as a new regulator of tolerance to IVM. Loss-of-function nhr-8(ok186) C. elegans mutants subjected to larval development assays and electropharyngeogram measurements, displayed hypersensitivity to IVM, and silencing of nhr-8 in IVM-resistant worms increased IVM efficacy. In addition, compared to wild-type worms, nhr-8 mutants under IVM selection pressure failed to acquire tolerance to the drug. In addition, IVM-hypersensitive nhr-8(ok186) worms displayed low transcript levels of several genes from the xenobiotic detoxification network and a concomitant low Pgp-mediated drug efflux activity. Interestingly, some pgp and cyp genes known to impact IVM tolerance in many nematode species, were down regulated in nhr-8 mutants and inversely upregulated in IVM-resistant worms. Moreover, pgp-6 overexpression in nhr-8(ok186) C. elegans increased tolerance to IVM. Importantly, NHR-8 function was rescued in nhr-8(ok186) C. elegans with the homolog of the parasitic nematode Haemonchus contortus, and silencing of Hco-nhr-8 by RNAi on L2 H. contortus larvae increased IVM susceptibility in both susceptible and resistant H. contortus isolates. Thus, our data show that NHR-8 controls the tolerance and development of resistance to IVM in C. elegans and the molecular basis for this relates to the NHR-8-mediated upregulation of IVM detoxification genes. Since our results show that Hco-nhr-8 functions similarly to Cel-nhr-8, this study helps to better understand mechanisms underlying failure in drug efficacy and open perspectives in finding new compounds with NHR-8 antagonist activity to potentiate IVM efficacy., Competing Interests: The authors have declared that no competing interests exist.

The relatively poor sensitivity is the main bottleneck restricting the application of microbial fuel cell biosensor (MFC-biosensor) for toxicity monitoring. Previous studies have shown that external resistance (R ext ) had an obvious effect on sensor sensitivity. However, these studies reported different results and the reason of this discrepancy was not clear. The objective of this research was to observe the effect of R ext on sensor sensitivity when detecting different types of pollutants and reveal its microbiological mechanism. Results demonstrated that the optimal R ext of MFC-biosensor varied with the type of pollutants. The optimal values for detecting avermectins, tetracyclines and heavy metals were 100 Ω, 330 Ω and 680 Ω, respectively. This discrepancy was mainly due to the visible differences in anodic microbial communities at different R ext settings. Both Azospirillum and Acinetobacter were susceptible to Cd and Pb, occuping 19.20% of the anodic microbial population in 680 Ω MFC-biosensor. Pseudomonas accounted for 10.73% in 330 Ω MFC-biosensor and possessed the sensitivity to tetracyclines. As for 100 Ω MFC-biosensor, the avermectin-intolerant Ocillibacter made up 2.55% of the anodic microbial community. This study indicated that the R ext of MFC-biosensor should be optimized according to the potential pollutants., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Avermectins, are pesticides of fermentation products with high efficacy and low toxicity and play important roles in food and agricultural product safety, animal and human health. Ivermectins, the derivatives of avermectins, are used to treat Onchocerciasis, also known as River Blindness. Thanks to these discoveries, more than 200 million Africans are lucky to be exempted from blindness. Great efforts have been made on better understanding of the microbial production system of avermectins through genetic engineering in China. Starting from scratch, Chinese avermectin industry has experienced a comprehensive technological innovation with significantly improved yield, titer and productivity of avermectins, and became the exclusive supplier for the global market. The success of avermectin industry innovation in China not only shed lights on the intelligent yield improvement of other microbial natural product drugs, but also gained wide international recognition. We provide here an overview of the discovery and development of basic and applied research of avermectins, especially historical evolution of avermectins made in China, which will benefit industrial development of microbial drugs in China.

While ~30% of the human genome encodes membrane proteins, only a handful of structures of membrane proteins have been resolved to high resolution. Here, we studied the structure of a member of the Cys-loop ligand gated ion channel protein superfamily of receptors, human type A γ 2 α 1 β 2 α 1 β 2 gamma amino butyric acid receptor complex in a lipid bilayer environment. Studying the correlation between the structure and function of the gamma amino butyric acid receptor may enhance our understanding of the molecular basis of ion channel dysfunctions linked with epilepsy, ataxia, migraine, schizophrenia and other neurodegenerative diseases. The structure of human γ 2 α 1 β 2 α 1 β 2 has been modeled based on the X-ray structure of the Caenorhabditis elegans glutamate-gated chloride channel via homology modeling. The template provided the first inhibitory channel structure for the Cys-loop superfamily of ligand-gated ion channels. The only available template structure before this glutamate-gated chloride channel was a cation selective channel which had very low sequence identity with gamma aminobutyric acid receptor. Here, our aim was to study the effect of structural corrections originating from modeling on a more reliable template structure. The homology model was analyzed for structural properties via a 100 ns molecular dynamics (MD) study. Due to the structural shifts and the removal of an open channel potentiator molecule, ivermectin, from the template structure, helical packing changes were observed in the transmembrane segment. Namely removal of ivermectin molecule caused a closure around the Leu 9 position along the ion channel. In terms of the structural shifts, there are three potential disulfide bridges between the M1 and M3 helices of the γ 2 and 2 α 1 subunits in the model. The effect of these disulfide bridges was investigated via monitoring the differences in root mean square fluctuations (RMSF) of individual amino acids and principal component analysis of the MD trajectory of the two homology models-one with the disulfide bridge and one with protonated Cys residues. In all subunit types, RMSF of the transmembrane domain helices are reduced in the presence of disulfide bridges. Additionally, loop A, loop F and loop C fluctuations were affected in the extracellular domain. In cross-correlation analysis of the trajectory, the two model structures displayed different coupling in between the M2-M3 linker region, protruding from the membrane, and the β1-β2/D loop and cys-loop regions in the extracellular domain. Correlations of the C loop, which collapses directly over the bound ligand molecule, were also affected by differences in the packing of transmembrane helices. Finally, more localized correlations were observed in the transmembrane helices when disulfide bridges were present in the model. The differences observed in this study suggest that dynamic coupling at the interface of extracellular and ion channel domains differs from the coupling introduced by disulfide bridges in the transmembrane region. We hope that this hypothesis will be tested experimentally in the near future.

Background: Root-knot nematode (Meloidogyne spp., RKN) causes a disease that significantly reduces the yield of greenhouse cucumber crops year after year. Chemical control based on a single pesticide is now unreliable mainly due to pest resistance. Fumigant and non-fumigant pesticide combinations can potentially result in effective and economic RKN control., Results: Combining the insecticide abamectin (ABM) with fumigants dazomet (DZ) or chloropicrin (CP) significantly extended the half-life of ABM by an average of about 1.68 and 1.56 times respectively in laboratory trials, and by an average of about 2.02 and 1.69 times respectively in greenhouse trials. Laboratory experiments indicated that all the low rate ABM combination treatments controlled RKN through a synergistic effect. ABM diffused into the nematode epidermis more rapidly when ABM was combined with DZ and CP, giving effective nematode control and an increase cucumber total yield, compared to the use of these products alone. ABM combined with CP or DZ produced significantly higher total cucumber yield than when these products were used alone., Conclusions: A low concentration of ABM combined with DZ in preference to CP would be an economic and practical way to control nematode and soilborne fungi in a greenhouse producing cucumbers., Competing Interests: The authors have declared that no competing interests exist.

Abamectin (ABM) is a widely utilized potent anthelmintic and insecticidal agent. In this study, we investigated the protective effects of caffeic acid phenethyl ester (CAPE) and betaine (BET) against ABM-induced hepatotoxicity and nephrotoxicity in rats. Forty rats were divided into five groups, receiving either oral saline solution (normal control), oral ABM at a dose of 2 mg/kg BW (1/5 LD50), CAPE (10 μmol/kg BW intraperitoneally) followed by ABM, or BET supplementation at a dose of 250 mg/kg BW followed by ABM administration, while group V rats received a combination of i.p. CAPE and oral BET in the same doses before receiving ABM. Biochemical analysis showed that ABM administration significantly (p < 0.05) increased serum levels of aminotransferases, alkaline phosphatase, lactate dehydrogenase, and cholesterol, as well as serum creatinine and urea. Compared to the control group, ABM-intoxicated rats had significantly (p < 0.05) higher tissue concentrations of nitric oxide and malondialdehyde, as well as lower tissue glutathione concentration, total antioxidant capacity, and antioxidant enzymatic activity (glutathione peroxidase, superoxide dismutase, and catalase). Histopathological examination of hepatic and renal tissues of ABM-intoxicated rats showed acute inflammatory and necrotic changes. Pretreatment with CAPE and/or BET reversed the biochemical and histopathological alterations of ABM on the liver and kidneys. Therefore, CAPE and BET (alone or in combination) could be promising protective agents against ABM-induced hepatotoxicity and nephrotoxicity. Future studies should confirm our findings and evaluate the other molecular effects are involved in the combination chemoprotection of CAPE and BET.

The deleterious effects of alcohol use disorders (AUDs) on human health have been documented worldwide. The enormous socioeconomic burden coupled with lack of efficacious pharmacotherapies underlies the need for improved treatment strategies. At present, there is a growing body of preclinical evidence that demonstrates the potential of avermectins [ivermectin (IVM), selamectin (SEL), abamectin (ABM), and moxidectin (MOX)] in treatment of AUDs. Avermectins are derived by fermentation of soil micro-organism, Streptomyces avermitilis, and have been extensively used for treatment of parasitic infections. From the mechanistic standpoint, avermectins are positive modulators of purinergic P2X4 receptors (P2X4Rs). P2X4Rs belong to P2X superfamily of cation-permeable ion channels gated by adenosine 5'-triphosphate (ATP). Building evidence has implicated a role for P2X4Rs in regulation of ethanol intake and that ethanol can inhibit ATP-gated currents in P2X4Rs. Investigations using recombinant cell models and animal models of alcohol drinking have reported that IVM, ABM, and MOX, but not SEL, were able to antagonize the inhibitory effects of ethanol on P2X4Rs in vitro and reduce ethanol intake in vivo. Furthermore, IVM was shown to reduce ethanol consumption via P2X4R potentiation in vivo, supporting the involvement of P2X4Rs in IVM's anti-alcohol effects and that P2X4Rs can be used as a platform for developing novel anti-alcohol compounds. Taken together, these findings support the utility of avermectins as a novel class of drug candidates for treatment of AUDs.

Aims: To determine the plasma disposition and concentrations of ivermectin (IVM) in eggs produced by laying hens following S/C, oral and I/V administration., Methods: Twenty-four laying hens, aged 37 weeks and weighing 1.73 (SD 0.12) kg were allocated to three groups of eight birds. The injectable formulation of IVM was administered either orally, S/C, or I/V, at a dose of 0.2 mg/kg liveweight, following dilution (1:5, v/v) with propylene glycol. Heparinised blood samples were collected at various times between 0.25 hours and 20 days after drug administration. Eggs produced by hens were also collected daily throughout the study period. Samples of plasma and homogenised egg were analysed using HPLC., Results: Maximum concentrations of IVM in plasma and mean residence time of IVM were lower after oral (10.2 (SD 7.2) ng/mL and 0.38 (SD 0.14) days, respectively) than after S/C (82.9 (SD 12.4) ng/mL and 1.05 (SD 0.24) days, respectively) administration (p<0.01). The time to maximum concentration and elimination half-life were shorter following oral (0.14 (SD 0.04) and 0.23 (SD 0.11) days, respectively) than S/C (0.25 (SD 0.00) and 1.45 (SD 0.45) days, respectively) administration (p<0.01). IVM was first detected in eggs 2 days after treatment in all groups and was detected until 8 days after oral and I/V administration, and until 15 days after S/C administration. Peak concentrations of IVM were 15.7, 23.3 and 1.9 µg/kg, observed 2, 5 and 4 days after I/V, S/C and oral administration, respectively., Conclusions and Clinical Relevance: The low plasma bioavailability of IVM observed after oral administration in laying hens could result in lower efficacy or subtherapeutic plasma concentrations, which may promote the development of parasitic drug resistance. Due to high IVM residues in eggs compared to the maximum residue limits for other food-producing animal species, a withdrawal period should be necessary for eggs after IVM treatment in laying hens.

In streptomycetes, autoregulators are important signaling compounds that trigger secondary metabolism, and they are regarded as Streptomyces hormones based on their extremely low effective concentrations (nM) and the involvement of specific receptor proteins. Our previous distribution study revealed that butenolide-type Streptomyces hormones, including avenolide, are a general class of signaling molecules in streptomycetes and that Streptomyces albus strain J1074 may produce butenolide-type Streptomyces hormones. Here, we describe metabolite profiling of a disruptant of the S. albus aco gene, which encodes a key biosynthetic enzyme for butenolide-type Streptomyces hormones, and identify four butenolide compounds from S. albus J1074 that show avenolide activity. The compounds structurally resemble avenolide and show different levels of avenolide activity. A dual-culture assay with imaging mass spectrometry (IMS) analysis for in vivo metabolic profiling demonstrated that the butenolide compounds of S. albus J1074 stimulate avermectin production in another Streptomyces species, Streptomyces avermitilis , illustrating the complex chemical interactions through interspecies signals in streptomycetes. IMPORTANCE Microorganisms produce external and internal signaling molecules to control their complex physiological traits. In actinomycetes, Streptomyces hormones are low-molecular-weight signals that are key to our understanding of the regulatory mechanisms of Streptomyces secondary metabolism. This study reveals that acyl coenzyme A (acyl-CoA) oxidase is a common and essential biosynthetic enzyme for butenolide-type Streptomyces hormones. Moreover, the diffusible butenolide compounds from a donor Streptomyces strain were recognized by the recipient Streptomyces strain of a different species, resulting in the initiation of secondary metabolism in the recipient. This is an interesting report on the chemical interaction between two different streptomycetes via Streptomyces hormones. Information on the metabolite network may provide useful hints not only to clarification of the regulatory mechanism of secondary metabolism, but also to understanding of the chemical communication among streptomycetes to control their physiological traits., (Copyright © 2018 American Society for Microbiology.)

Haemonchus contortus, one of the most economically important parasites of small ruminants, has become resistant to the anthelmintic ivermectin. Deciphering the role of P-glycoproteins in ivermectin resistance is desirable for understanding and overcoming this resistance. In the model nematode, Caenorhabditis elegans, P-glycoprotein-13 is expressed in the amphids, important neuronal structures for ivermectin activity. We have focused on its ortholog in the parasite, Hco-Pgp-13. A 3D model of Hco-Pgp-13, presenting an open inward-facing conformation, has been constructed by homology with the Cel-Pgp-1 crystal structure. In silico docking calculations predicted high affinity binding of ivermectin and actinomycin D to the inner chamber of the protein. Following in vitro expression, we showed that ivermectin and actinomycin D modulated Hco-Pgp-13 ATPase activity with high affinity. Finally, we found in vivo Hco-Pgp-13 localization in epithelial, pharyngeal and neuronal tissues. Taken together, these data suggest a role for Hco-Pgp-13 in ivermectin transport, which could contribute to anthelmintic resistance., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

To avoid potential systematical toxicity, solid lipid nanoparticles (SLNs) were prepared as a vehicle for transdermal delivery of ivermectin (IVM) using hot homogenisation followed by ultrasonic method. The as-prepared SLNs were approximately spherical shape with good stability. IVM was encapsulated in amorphous form within SLNs and displayed prolonged release from SLNs without burst release due to high encapsulation efficiency (EE). The cumulative permeation of IVM across excised rat skin from SLNs was significantly increased compared to the ivermection suspension. These results indicated that the proposed SLNs can be considered as an efficient carrier for dermal delivery of IVM to effectively treat scabies.

Avermectin produced by Streptomyces avermitilis is an anti-nematodal agent against the pine wood nematode Bursaphelenchus xylophilus. However, its potential usage is limited by its poor water solubility. For this reason, continuous efforts are underway to produce new derivatives that are more water soluble. Here, the enzymatic glycosylation of avermectin was catalyzed by uridine diphosphate (UDP)-glycosyltransferase from Bacillus licheniformis with various UDP sugars. As a result, the following four avermectin B 1a glycosides were produced: avermectin B 1a 4″-β-D-glucoside, avermectin B 1a 4″-β-D-galactoside, avermectin B 1a 4″-β-L-fucoside, and avermectin B 1a 4″-β-2-deoxy-D-glucoside. The avermectin B 1a glycosides were structurally analyzed based on HR-ESI MS and 1D and 2D nuclear magnetic resonance spectra, and the anti-nematodal effect of avermectin B 1a 4″-β-D-glucoside was found to exhibit the highest activity (IC 50  = 0.23 μM), which was approximately 32 times greater than that of avermectin B 1a (IC 50  = 7.30 μM), followed by avermectin B 1a 4″-β-2-deoxy-D-glucoside (IC 50  = 0.69 μM), avermectin B 1a 4″-β-L-fucoside (IC 50  = 0.89 μM), and avermectin B 1a 4″-β-D-galactoside (IC 50  = 1.07 μM). These results show that glycosylation of avermectin B 1a effectively enhances its in vitro anti-nematodal activity and that avermectin glycosides can be further applied for treating infestations of the pine wood nematode B. xylophilus.

Background: The mosquito resistance to the insecticides threatens malaria control efforts, potentially becoming a major public health issue. Alternative methods like ivermectin (IVM) administration to humans has been suggested as a possible vector control to reduce Plasmodium transmission. Anopheles aquasalis and Anopheles darlingi are competent vectors for Plasmodium vivax, and they have been responsible for various malaria outbreaks in the coast of Brazil and the Amazon Region of South America., Methods: To determine the IVM susceptibility against P. vivax in An. aquasalis and An. darlingi, ivermectin were mixed in P. vivax infected blood: (1) Powdered IVM at four concentrations (0, 5, 10, 20 or 40 ng/mL). (2) Plasma (0 hours, 4 hours, 1 day, 5, 10 and 14 days) was collected from healthy volunteers after to administer a single oral dose of IVM (200 μg/kg) (3) Mosquitoes infected with P. vivax and after 4 days was provided with IVM plasma collected 4 hours post-treatment (4) P. vivax-infected patients were treated with various combinations of IVM, chloroquine, and primaquine and plasma or whole blood was collected at 4 hours. Seven days after the infective blood meal, mosquitoes were dissected to evaluate oocyst presence. Additionally, the ex vivo effects of IVM against asexual blood-stage P. vivax was evaluated., Results: IVM significantly reduced the prevalence of An. aquasalis that developed oocysts in 10 to 40 ng/mL pIVM concentrations and plasma 4 hours, 1 day and 5 days. In An. darlingi to 4 hours and 1 day. The An. aquasalis mortality was expressively increased in pIVM (40ng/mL) and plasma 4 hours, 1, 5 10 and 14 days post-intake drug and in An. darlingi only to 4 hours and 1 day. The double fed meal with mIVM by the mosquitoes has a considerable impact on the proportion of infected mosquitoes for 7 days post-feeding. The oocyst infection prevalence and intensity were notably reduced when mosquitoes ingested blood from P. vivax patients that ingested IVM+CQ, PQ+CQ and IVM+PQ+CQ. P. vivax asexual development was considerably inhibited by mIVM at four-fold dilutions., Conclusion: In conclusion, whole blood spiked with IVM reduced the infection rate of P. vivax in An. aquasalis and An. darlingi, and increased the mortality of mosquitoes. Plasma from healthy volunteers after IVM administration affect asexual P. vivax development. These findings support that ivermectin may be used to decrease P. vivax transmission.

We previously observed that ivermectin-induced detoxification genes, including ATP binding cassette transporter C4 (PhABCC4) and cytochrome P450 6CJ1 (CYP6CJ1) were identified from body lice following a brief exposure to a sublethal dose of ivermectin using a non-invasive induction assay. In this current study, the functional properties of PhABCC4 and CYP6CJ1 were investigated after expression in either X. laevis oocytes or using a baculovirus expression system, respectively. Efflux of [ 3 H]-9-(2-phosphonomethoxyethyl) adenine ([ 3 H]-PMEA), a known ABCC4 substrate in humans, was detected from PhABCC4 cRNA-injected oocytes by liquid scintillation spectrophotometric analysis and PhABCC4 expression in oocytes was confirmed using ABC transporter inhibitors. Efflux was also determined to be ATP-dependent. Using a variety of insecticides in a competition assay, only co-injection of ivermectin and dichlorodiphenyltrichloroethane led to decreased efflux of [ 3 H]-PMEA. PhABCC4-expressing oocytes also directly effluxed [ 3 H]-ivermectin, which increased over time. In addition, ivermectin appeared to be oxidatively metabolized and/or sequestered, although at low levels, following functional expression of CYP6CJ1 along with cytochrome P450 reductase in Sf9 cells. Our study suggests that PhABCC4 and perhaps CYP6CJ1 are involved in the Phase III and Phase I xenobiotic metabolism of ivermectin, respectively, and may play an important role in the evolution of ivermectin resistance in lice and other insects as field selection occurs., (© 2017 The Royal Entomological Society.)

Avermectins, produced by Streptomyces avermitilis, are important antiparasitic agents. The use of traditional microbial breeding methods for this organism has been limited by the low-throughput shake flask-based screening process. The unique growth cycle of actinomycetes makes the establishment of a reliable high-throughput screening (HTS) process difficult. To enhance the efficiency of screening strains with high yields of avermectin, a HTS process aided by fluorescence-activated cell sorting (FACS) was established. Four different spore solutions were investigated for maintaining a relatively high viability of spores. Propidium iodide (PI) and fluorescein diacetate (FDA) were used to discriminate between dead and live spores using the FACS system. Spores stained with 7-μg/mL PI and 15-μg/mL FDA at 4 °C in the dark for 30 min resulted in optimum sorting. Spores were treated by atmospheric and room temperature plasma (ARTP). Single live spores were sorted and sprayed into 96-well microtiter plates containing 50 μL of solid agar culture medium. Solid-liquid combinatorial microculture was used for high-throughput avermectin culture. A high-titer avermectin producer (G9) was obtained from 5760 mutants after mutagenesis and HTS. Compared with the original strain, the titer was improved by 18.9% on flask culture and 20.6% on fermenter, respectively. The HTS process established in this study could easily be transferred to other similar target products produced by actinomycetes.

Microbial strains are amazingly clever by homeostasis of their own survival and optimization for the overproduction of a desired phenotype, for example drugable secondary metabolites through coordination of key genes overexpression and media optimizations. Besides their pesticide activities, avermectins (AVMs) are identified as potent antibiotic agents for a wide range of drug-resistant pathogens by a high-throughput synergy screening strategy. To rewire the genetic circuitry controlling low yields, we summarized the work on balancing the biological chassis with functional parts, and optimized their dynamical process, as well as predicted favorable effective overproduction of AVMs by 5Ms strategy. AVMs are exclusively made in China now and intelligences learned from the success of AVMs will help transform microbes into a true power-house of innovation., (Copyright © 2017. Published by Elsevier Ltd.)

Fluralaner (Bravecto) is a recently marketed isoxazoline ectoparasiticide. This compound potently inhibits GABA-gated chloride channels (GABACls) and less potently glutamate-gated chloride channels (GluCls) in insects. The mechanism underlying this selectivity is unknown. Therefore, we sought to identify the amino acid residues causing the low potency of fluralaner toward GluCls. We examined the fluralaner sensitivity of mutant housefly ( Musca domestica ) GluCls in which amino acid residues in the transmembrane subunit interface were replaced with the positionally equivalent amino acids of Musca GABACls. Of these amino acids, substitution of an amino acid (Leu315) in the third transmembrane region (TM3) with an aromatic amino acid dramatically enhanced the potency of fluralaner in the GluCls. In stark contrast to the enhancement of fluralaner potency, this mutation eliminated the activation of currents and the potentiation but not the antagonism of glutamate responses that are otherwise all elicited by the macrolide parasiticide ivermectin (IVM). Our findings indicate that the amino acid Leu315 in Musca GluCls plays significant roles in determining the selectivity of fluralaner and IVM for these channels. Given the high sequence similarity of TM3, this may hold true more widely for the GluCls and GABACls of other insect species., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

We examined the removal of abamectin by the electro-Fenton (EF) process and the feasibility of biological treatment after degradation. The effect of the operating parameters showed that abamectin (Aba) degradation was enhanced with increasing temperature. Response surface analysis of the central composite design led to the following optimal conditions for the abatement of chemical oxygen demand: 45.5 °C, 5 mg L -1 , 150 mA, and 0.15 mmol L -1 for the temperature, initial Aba concentration, current intensity, and catalyst concentration, respectively. Under these conditions, 68.01% of the organic matter was removed and 94% of Aba was degraded after 5 h and 20 min of electrolysis, respectively. A biodegradability test, which was performed on a solution electrolyzed at 47 °C, 9 mg L -1 , 150 mA, and 0.15 mmol L -1 , confirms that the ratio of biological oxygen demand/chemical oxygen demand increased appreciably from 0.0584 to 0.64 after 5 h of electrolysis. This increased ratio is slightly above the limit of biodegradability (0.4). These results show the relevance of the EF process and its effectiveness for abamectin degradation. We conclude that biological treatment can be combined with the EF process for total mineralization.

Objective: To improve the production of short branched-chain acyl-CoAs for avermectin biosynthesis, the functional expression of the branched chain α-keto acid dehydrogenase complex (BKDH) from Streptomyces avermitilis was systematically optimized by selectively regulating individual subunit expression in Escherichia coli., Results: Functional expression of the BKDH complex was achieved by independent and selective optimization of individual subunit genes of the complex. Codon optimization significantly improved the expression of complex component proteins BkdH and LpdA1 but expression of BkdF and BkdG depended on coexpression of the bkdH gene. The optimized BKDH complex supplied sufficient short branched-chain acyl-CoA to synthesize phlorisovalerophenone, a key intermediate in bitter acid (humulone) synthesis. We also developed a novel p15A origin-derived high-copy-number vector system for expression; the yield of PIVP was 350 ng/mOD., Conclusion: Through optimization strategies, we obtained stable, functional expression of the BKDH complex in E. coli, which could be applied in the heterologous production of numerous high-value-added chemicals, especially polyketides.

The pH-sensitive chloride channels (pHCls) are broadly expressed in insects, but little is known about their physiologic role, diversity, and sensitivity to insecticides acting on relevant chloride channels. Here we have sequenced 50 transcripts of the pHCl-1 gene from the brain, third thoracic ganglion (T3G), and midgut of larvae of silkworm Bombyx mori It was found that >50 variants were expressed with distinct splicing in the T3G compared with the brain and midgut. Of the variants detected, variant 9, which was expressed most abundantly in the larvae, was reconstituted in Xenopus laevis oocytes to characterize its pH and ivermectin sensitivity. Variant 9 formed a functional pHCl with half-maximal activation at a pH of 7.87, and was activated by ivermectin irrespective of the extracellular pH. This was in contrast to variant 1, which was activated more profoundly at acidic rather than basic pH. To identify a key determinant for such differential ivermectin sensitivity, different amino acids in variants 1 and 9 were swapped, and the effects of the mutations on ivermectin sensitivity were investigated. The V275S mutation of variant 1 enhanced ivermectin sensitivity, whereas the S275V mutation of variant 9 caused a reduction in sensitivity. In homology models of the Bombyx pHCls, Val275 of variant 1 interacted more strongly with Ala273 than Ser275 of variant 9 at the channel gate. This interaction is likely to prevent ivermectin-induced opening of the channel, accounting, at least partially, for the differential macrolide action on the two variants., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

The P2X4 receptor (P2X4R) is a member of a family of purinergic channels activated by extracellular ATP through three orthosteric binding sites and allosterically regulated by ivermectin (IVM), a broad-spectrum antiparasitic agent. Treatment with IVM increases the efficacy of ATP to activate P2X4R, slows both receptor desensitization during sustained ATP application and receptor deactivation after ATP washout, and makes the receptor pore permeable to NMDG+, a large organic cation. Previously, we developed a Markov model based on the presence of one IVM binding site, which described some effects of IVM on rat P2X4R. Here we present two novel models, both with three IVM binding sites. The simpler one-layer model can reproduce many of the observed time series of evoked currents, but does not capture well the short time scales of activation, desensitization, and deactivation. A more complex two-layer model can reproduce the transient changes in desensitization observed upon IVM application, the significant increase in ATP-induced current amplitudes at low IVM concentrations, and the modest increase in the unitary conductance. In addition, the two-layer model suggests that this receptor can exist in a deeply inactivated state, not responsive to ATP, and that its desensitization rate can be altered by each of the three IVM binding sites. In summary, this study provides a detailed analysis of P2X4R kinetics and elucidates the orthosteric and allosteric mechanisms regulating its channel gating.

Ivermectin acts as a positive allosteric modulator of several Cys-loop receptors including the glutamate-gated chloride channels (GluCls), γ-aminobutyric acid receptors (GABA A Rs), glycine receptors (GlyRs), and neuronal α7-nicotinic receptors (α7 nAChRs). The crystal structure of Caenorhabditis elegans GluCl complexed with ivermectin revealed the details of its ivermectin binding site. Although the electron microscopy structure of zebrafish GlyRα1 complexed with ivermectin demonstrated a similar binding orientation, detailed structural information on the ivermectin binding and pore opening for Cys-loop receptors in vertebrates has been elusive. Here we present the crystal structures of human GlyRα3 in complex with ivermectin at 2.85 and 3.08 Å resolution. Our structures allow us to explore in detail the molecular recognition of ivermectin by GlyRs, GABA A Rs, and α7 nAChRs. Comparisons with previous structures reveal how the ivermectin binding expands the ion channel pore. Our results hold promise in structure-based design of GlyR modulators for the treatment of neuropathic pain., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Drug interactions between antiretroviral drugs (ARVs) and anthelminthic drugs, ivermectin (IVM) and praziquantel (PZQ) were assessed by investigating their permeation through the Caco-2 cell monolayers in a transwell. The impact of anthelminthics on the transport of ARVs was determined by assessing the apical to basolateral (AP → BL) [passive] and basolateral to apical (BL → AP) [efflux] directions alone, and in presence of an anthelminthic. The reverse was conducted for the assessment of the influence of ARVs on anthelminthics., Methods: Samples from the AP and BL compartments were taken at 60, 120, 180 and 240 min and quantified either by HPLC or radiolabeled assay using a liquid scintillating counter for the respective drugs. Transepithelial resistance (TEER) was used to assess the integrity of the monolayers. The amount of compound transported per second (apparent permeability, Papp) was calculated for both AP to BL (Papp AtoB ), and BL to AP (Papp BtoA ) movements. Samples collected after 60 min were used to determine the efflux ratio (ER), quotient of secretory permeability and absorptive permeability (PappBL-AP/PappAP-BL). The reverse, (PappAP-BL/PappBL-AP) constituted the uptake ratio. The impact of SQV, EFV and NVP on the transport of both IVM and PZQ were investigated. The effect of LPV on the transport of IVM was also determined. The influence of IVM on the transport of SQV, NVP, LPV and EFV; as well as the effect PZQ on the transport of SQV of was also investigated, and a two-tailed p value of <0.05 was considered significant., Results: IVM significantly inhibited the efflux transport (BL → AP movement) of LPV (ER; 6.7 vs. 0.8, p = 0.0038) and SQV (ER; 3.1 vs. 1.2 p = 0.00328); and increased the efflux transport of EFV (ER; 0.7 vs. 0.9, p = 0.031) suggesting the possibility of drug transporter mediated interactions between the two drugs. NVP increased the efflux transport of IVM (ER; 0.8 vs. 1.8, p = 0.0094)., Conclusions: The study provides in vitro evidence of potential interactions between IVM, an anthelminthic drug with antiretroviral drugs; LPV, SQV, NVP and EFV. Further investigations should be conducted to investigate the possibility of in vivo interactions.

It was found that S-adenosylmethionine (SAM) could effectively improve avermectin titer with 30-60 μg/mL addition to FH medium. To clearly elucidate the mechanism of SAM on intracellular metabolites of Streptomyces avermitilis, a GC-MS-based comparative metabolomics approach was carried out. First, 230 intracellular metabolites were identified and 14 of them remarkably influenced avermectin biosynthesis were discriminative biomarkers between non-SAM groups and SAM-treated groups by principal components analysis (PCA) and partial least squares (PLS). Based on further key metabolic pathway analyses, these biomarkers, such as glucose, oxaloacetic acid, fatty acids (in soybean oil), threonine, valine, and leucine, were identified as potentially beneficial precursors and added in medium. Compared with single-precursor feeding, the combined feeding of the precursors and SAM markedly increased the avermectin titer. The co-feeding approach not only directly verified our hypothesis on the mechanism of SAM by comparative metabolomics, but also provided a novel strategy to increase avermectin production.

Helminth infections are widespread in the poultry industry. There is evidence of extra-label use of some drugs, such as ivermectin (IVM), in broiler poultry. Pharmacokinetic and residual studies of IVM in poultry, however, are rather scarce. Our aim was to determine time restrictions for broiler chickens fed with balanced feed mixed with IVM for 21 days, and thus achieve acceptable residual levels for consumption as established by the European Union. Sixty 1-day-old chicks were fed with food supplemented with IVM at 5 mg kg -1 feed for 21 days. Groups of six treated animals were sacrificed at 0, 1, 2, 4, 8, 10, 15, 20 and 28 days after treatment. Liver, skin/fat, kidney and muscle samples were obtained. IVM were determined by liquid chromatography with fluorescence detection after automatic solid-phase extraction with SPE C 18 cartridges. The highest concentrations were measured in the liver, which is logical given that IVM is a drug that undergoes extensive hepatic metabolism. The optimal withdrawal time for edible tissues of these animals to stay within the permitted residual levels were: 12 days for liver, 8 days for skin/fat, 0 days for muscle and 10 days for kidney.

A spectrophotometric screening method for avermectin oxidizing microbes by determination of 4″-oxo-avermectin was established based on the reaction between 4″-oxo-avermectin and 2,4-dinitrophenylhydrazine. Combined with a gradient HPLC assay, microorganisms capable of regioselectively oxidizing avermectin to 4″-oxo-avermectin were successfully obtained by this method., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The regulatory role of redox-sensing regulator Rex was investigated in Streptomyces avermitilis. Eleven genes/operons were demonstrated to be directly regulated by Rex; these genes/operons are involved in aerobic metabolism, morphological differentiation, and secondary metabolism. Rex represses transcription of target genes/operons by binding to Rex operator (ROP) sequences in the promoter regions. NADH reduces DNA-binding activity of Rex to target promoters, while NAD + competitively binds to Rex and modulates its DNA-binding activity. Rex plays an essential regulatory role in aerobic metabolism by controlling expression of the respiratory genes atpIBEFHAGDC, cydA1B1CD, nuoA1-N1, rex-hemAC1DB, hppA, and ndh2. Rex also regulates morphological differentiation by repressing expression of wblE, which encodes a putative WhiB-family transcriptional regulator. A rex-deletion mutant (Drex) showed higher avermectin production than the wild-type strain ATCC31267, and was more tolerant of oxygen limitation conditions in regard to avermectin production.