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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).)

Base editing (BE) faces protospacer adjacent motif (PAM) constraints and off-target effects in both eukaryotes and prokaryotes. For Streptomyces, renowned as one of the most prolific bacterial producers of antibiotics, the challenges are more pronounced due to its diverse genomic content and high GC content. Here, we develop a base editor named eSCBE3-NG-Hypa, tailored with both high efficiency and -fidelity for Streptomyces. Of note, eSCBE3-NG-Hypa recognizes NG PAM and exhibits high activity at challenging sites with high GC content or GC motifs, while displaying minimal off-target effects. To illustrate its practicability, we employ eSCBE3-NG-Hypa to achieve precise key amino acid conversion of the dehydratase (DH) domains within the modular polyketide synthase (PKS) responsible for the insecticide avermectins biosynthesis, achieving domains inactivation. The resulting DH-inactivated mutants, while ceasing avermectins production, produce a high yield of oligomycin, indicating competitive relationships among multiple biosynthetic gene clusters (BGCs) in Streptomyces avermitilis. Leveraging this insight, we use eSCBE3-NG-Hypa to introduce premature stop codons into competitor gene cluster of ave in an industrial S. avermitilis, with the mutant Δolm exhibiting the highest 4.45-fold increase in avermectin B1a compared to the control. This work provides a potent tool for modifying biosynthetic pathways and advancing metabolic engineering in Streptomyces., (© 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).)

The ivermectin is a potent nematocide and insecticide, which has low toxicity for humans and domestic animals, but due to low biotransformation, it can be dangerous for non-target organisms. The recent determination of ivermectin absorption and accumulation in tissues of higher plants and multiple shreds of evidence of its negative impact on plant physiology provide a basis for the search for ivermectin's molecular targets and mechanisms of action in plant cells. In this research, for the first time, the ivermectin effect on microtubules of Arabidopsis thaliana cells was studied. It was revealed that ivermectin (250 μg mL -1 ) disrupts the microtubule network, induces the loss of microtubule orientation, leads to microtubule curvature and shrinkage, and their longitudinal and cross-linked bundling in various cells of A. thaliana primary roots. Further, the previously proposed binding of ivermectin to the β1-tubulin taxane site was developed and confirmed using molecular dynamics simulations of ivermectin complexes with Haemonchus contortus and A. thaliana β1-tubulins. It was predicted that similar to other microtubule stabilizing agents ivermectin binding causes M-loop stabilization in both H. contortus and A. thaliana β-tubulin, which leads to the enhancement of lateral contacts between subunits of adjacent protofilaments preventing microtubule depolymerization., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Ivermectin (IVM) is a dewormer commonly utilized in animal farming. Nevertheless, there is a deficiency of research on the bioecotoxicity of IVM in soil. In this study, earthworms were utilized as test animals to investigate the ecotoxicological impacts of IVM. The experiment lasted 28 days and involved adding varied doses of IVM to a culture substrate of soil mixed with cow dung and feeding it to earthworms. The experiment entailed recording earthworm weight, number of earthworm cocoons, histological damage, oxidative stress indicators, and gene expression levels. The analysis results showed that earthworm growth and reproduction were hampered by IVM. Moreover, pathological damage to the earthworms increased with increasing IVM concentration, which caused increased oxidative damage to the earthworms. These findings offer a summary of the impact of IVM on earthworms and a reference point for future research examining the ecological implications of IVM., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Importance: Dengue virus (DENV) is a major human pathogen that can cause hemorrhagic fever and shock syndrome. One important factor of DENV pathogenicity is non-structural protein 1 (NS1), a glycoprotein that is secreted from infected cells. Here we study the mode of action of the widely used drug ivermectin, used to treat parasitic infections and recently shown to lower NS1 blood levels in DENV-infected patients. We found that ivermectin blocks the nuclear transport of transcription factors required for the expression of chaperones that support the folding and secretion of glycoproteins, including NS1. Impairing nuclear transport of these transcription factors by ivermectin or depleting them from infected cells dampens NS1 folding and thus its secretion. These results reveal a novel mode of action of ivermectin that might apply to other flaviviruses as well., Competing Interests: The authors declare no conflict of interest.

Controlling Rhipicephalus microplus is among the most significant challenges for livestock production worldwide. The indiscriminate use of acaricides stimulates the selection of resistant tick populations and is therefore ineffective. Understanding the molecular foundations of resistance could help inform the search for new alternatives for tick control. Although the ovary has been suggested as a relevant target organ for tick control, there are few existing studies that focus on tick ovarian tissue. Therefore, we conducted a comparative proteomic analysis on ovaries of R. microplus strains with differential resistance to ivermectin. In resistant ticks, we observed the over-accumulation of proteins involved in several biological processes, including translation, proteolysis, transport, cellular organization, differentiation, and xenobiotic detoxification. We also observed the accumulation of many structural and extracellular proteins such as papilin-like protein, which glycosylation increase its stability-based molecular modeling. Therefore, we propose that ovaries of ivermectin-resistant ticks overcome the negative impact of ivermectin through the activation of detoxification mechanisms and structural proteins associated with the remodeling of the ovary's extracellular matrix. SIGNIFICANCE: Understanding the molecular foundation of ivermectin resistance in Rhipicephalus microplus represents an essential step in cattle farming, which could provide clues and alternatives for tick control. Excessive use of chemicals like ivermectin allows the generation of resistant tick strains in different countries. However, limited molecular information is available concerning the tick's resistance to ivermectin. Detailed proteomics scrutiny in various tick organs will provide more comprehensive molecular information. Thus, we conducted an ovary comparative proteomic-based TMT-SPS-MS3 approach. We highlight in ivermectin-resistant ticks the over-accumulation of structural proteins and enzymes connected to detoxification mechanisms., Competing Interests: Declaration of Competing Interest All the authors of this study attest to not having any conflict of interest in publishing this manuscript., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

Sea louse ectoparasitosis is a major threat to fish aquaculture. Avermectins such as ivermectin and emamectin have been effectively used against sea louse infestation, but the emergence of resistance has limited their use. A better understanding of the molecular targets of avermectins is essential to the development of novel treatment strategies or new, more effective drugs. Avermectins are known to act by inhibiting neurotransmission through allosteric activation of glutamate-gated chloride channels (GluCls). We have investigated the GluCl subunit present in Caligus rogercresseyi, a sea louse affecting aquaculture in the Southern hemisphere. We identify four new subunits, CrGluCl-B to CrGluCl-E, and characterise them functionally. CrGluCl-A (previously reported as CrGluClα), CrGluCl-B and CrGluCl-C all function as glutamate channel receptors with different sensitivities to the agonist, but in contrast to subunit -A and -C, CrGluCl-B is not activated by ivermectin but is rather antagonised by the drug. CrGluCl-D channel appears active in the absence of any stimulation by glutamate or ivermectin and CrGluCl-E does not exhibit any activity. Notably, the expression of CrGluCl-B with either -A or -C subunits gives rise to receptors unresponsive to ivermectin and showing altered response to glutamate, suggesting that coexpression has led to the preferential formation of heteromers to which the presence of CrGluCl-B confers the property of ivermectin-activation refractoriness. Furthermore, there was evidence for heteromer formation with novel properties only when coexpressing pairs E/C and D/B CrGluCl subtypes. Site-directed mutagenesis shows that three transmembrane domain residues contribute to the lack of activation by ivermectin, most crucially Gln 15' in M2, with mutation Q15'T (the residue present in ivermectin-activated subunits A and C) conferring ivermectin activation to CrGluCl-B. The differential response to avermectin of these Caligus rogercresseyi GluClsubunits, which are highly conserved in the Northern hemisphere sea louse Lepeophtheirus salmonis, could have an influence on the response of these parasites to treatment with macrocyclic lactones. They could serve as molecular markers to assess susceptibility to existing treatments and might be useful molecular targets in the search for novel antiparasitic drugs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Tribiños 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.)

Avermectin is a widely used insecticide, and it is mainly effective against animal parasites and insects. Given its extensive use in agriculture, a large amount of avermectin is accumulated in natural waters. Avermectin is a neurotoxin that affects the autonomous behavior of zebrafish and inhibits neurological responses in invertebrates via GABA-chloride channels. In this study, we used zebrafish as a model organism to explore the lethal teratogenic effects of different avermectin concentrations. We found that 50-μg/L avermectin could cause significant malformation abnormalities during the development of zebrafish heart, changes in heart rate, and significant reduction in hatching rate and body length. Transcriptome data revealed that 499 genes were upregulated and 877 genes were downregulated at 72 h post-fertilization (hpf), whereas 1805 genes were upregulated and 836 genes were downregulated at 120 hpf. According to gene ontology (GO) enrichment analysis, avermectin affected cardiac circulation and myocardial fiber development. KEGG analysis revealed that avermectin treatment significantly altered the activity of signal pathways associated with cardiac rhythm and vascular smooth muscle contraction. The main target of avermectin was identified as the heart, as it affected heart development and function by altering cardiac-related gene expression that led to a heart defect phenotype. Our findings indicate that developing zebrafish are sensitive to avermectin, which targets the heart., Competing Interests: Declaration of competing interest The authors have no competing interests to declare. We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Spin label electron paramagnetic resonance (EPR) spectroscopy was used to study the mechanisms of action of ivermectin and curcumin against Leishmania (L.) amazonensis promastigotes. EPR spectra showed that treatment of the parasites with both compounds results in plasma membrane rigidity due to oxidative processes. With the IC 50 and EPR measurements for assays using different parasite concentrations, estimations could be made for the membrane-water partition coefficient (K M/W ), and the concentration of the compound in the membrane (c m50 ) and in the aqueous phase (c w50 ), which inhibits cell growth by 50%. The K M/W values indicated that ivermectin has a greater affinity than curcumin for the parasite membrane. Therefore, the activity of ivermectin was higher for experiments with low cell concentrations, but for concentrations greater than 1.5 × 10 8 parasites/mL the compounds did not show significantly different results. The c m50 values indicated that the concentration of compound in the membrane leading to growth inhibition or membrane alteration is approximately 1 M for both ivermectin and curcumin. This high membrane concentration suggests that many ivermectin molecules per chlorine channel are needed to cause an increase in chlorine ion influx., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The number of reported macrocyclic lactones (ML) resistance cases across all livestock hosts is steadily increasing. Different studies in the parasitic nematode Haemonchus contortus assume the participation of cytochrome P450s (Cyps) enzymes in ML resistance. Still, functional data about their individual contribution to resistance or substrate specificity is missing. Via microinjection, transgenic Caenorhabditis elegans expressing HCON&#95;00141052 ( transgene-Hco-cyp-13A11 ) from extrachromosomal arrays were generated. After 24 h of exposure to different concentrations of ivermectin (IVM), ivermectin aglycone (IVMa), selamectin (SEL), doramectin (DRM), eprinomectin (EPR), and moxidectin (MOX), motility assays were performed to determine the impact of the H. contortus Cyp to the susceptibility of the worms against each ML. While transgene - Hco-cyp-13A11 significantly decreased susceptibility to IVM (four-fold), IVMa (2-fold), and SEL (3-fold), a slight effect for DRM and no effect for MOX, and EPR was observed. This substrate specificity of Hco-cyp-13A11 could not be explained by molecular modeling and docking studies. Hco-Cyp-13A11 molecular models were obtained for alleles from isolates with different resistance statuses. Although 14 amino acid polymorphisms were detected, none was resistance specific. In conclusion, Hco-cyp-13A11 decreased IVM, IVMa, and SEL susceptibility to a different extent, but its potential impact on ML resistance is not driven by polymorphisms.

The antiparasitic drug ivermectin plays an essential role in human and animal health globally. However, ivermectin resistance is widespread in veterinary helminths and there are growing concerns of sub-optimal responses to treatment in related helminths of humans. Despite decades of research, the genetic mechanisms underlying ivermectin resistance are poorly understood in parasitic helminths. This reflects significant uncertainty regarding the mode of action of ivermectin in parasitic helminths, and the genetic complexity of these organisms; parasitic helminths have large, rapidly evolving genomes and differences in evolutionary history and genetic background can confound comparisons between resistant and susceptible populations. We undertook a controlled genetic cross of a multi-drug resistant and a susceptible reference isolate of Haemonchus contortus, an economically important gastrointestinal nematode of sheep, and ivermectin-selected the F2 population for comparison with an untreated F2 control. RNA-seq analyses of male and female adults of all populations identified high transcriptomic differentiation between parental isolates, which was significantly reduced in the F2, allowing differences associated specifically with ivermectin resistance to be identified. In all resistant populations, there was constitutive upregulation of a single gene, HCON&#95;00155390:cky-1, a putative pharyngeal-expressed transcription factor, in a narrow locus on chromosome V previously shown to be under ivermectin selection. In addition, we detected sex-specific differences in gene expression between resistant and susceptible populations, including constitutive upregulation of a P-glycoprotein, HCON&#95;00162780:pgp-11, in resistant males only. After ivermectin selection, we identified differential expression of genes with roles in neuronal function and chloride homeostasis, which is consistent with an adaptive response to ivermectin-induced hyperpolarisation of neuromuscular cells. Overall, we show the utility of a genetic cross to identify differences in gene expression that are specific to ivermectin selection and provide a framework to better understand ivermectin resistance and response to treatment in parasitic helminths., Competing Interests: The authors have declared that no competing interests exist.

Zinc is an essential cofactor for many metal enzymes and transcription regulators. Zn 2+ availability has long been known to affect antibiotic production and morphological differentiation of Streptomyces species. However, the molecular mechanism whereby zinc regulates these processes remains unclear. We investigated the regulatory roles of the zinc-sensing regulator Zur in Streptomyces avermitilis. Our findings demonstrate that Zur plays an essential role in maintaining zinc homeostasis by repressing the expression of the zinc uptake system ZnuACB and alternative non-zinc-binding ribosomal proteins and promoting the expression of zinc exporter ZitB. Deletion of the zur gene resulted in decreased production of avermectin and oligomycin and delayed morphological differentiation, and these parameters were restored close to wild-type levels in a zur -complemented strain. Zur bound specifically to Zur box in the promoter regions of avermectin pathway-specific activator gene aveR , oligomycin polyketide synthase gene olmA1 , and filipin biosynthetic pathway-specific regulatory genes pteR and pteF . Analyses by reverse transcription quantitative PCR and luciferase reporter systems indicated that Zur directly activates the transcription of these genes, i.e., that Zur directly activates biosynthesis of avermectin and oligomycin. Zur positively regulated morphological development by repressing the transcription of differentiation-related genes ssgB and minD2 . Our findings, taken together, demonstrate that Zur in S. avermitilis directly controls zinc homeostasis, biosynthesis of avermectin and oligomycin, and morphological differentiation. IMPORTANCE Biosynthesis of secondary metabolites and morphological differentiation in bacteria are affected by environmental signals. The molecular mechanisms whereby zinc availability affects secondary metabolism and morphological differentiation remain poorly understood. We identified several new target genes of the zinc response regulator Zur in Streptomyces avermitilis, the industrial producer of avermectin. Zur was found to directly and positively control avermectin production, oligomycin production, and morphological differentiation in response to extracellular Zn 2+ levels. Our findings clarify the regulatory functions of Zur in Streptomyces , which involve linking environmental Zn 2+ status with control of antibiotic biosynthetic pathways and morphological differentiation.

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 novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still has serious negative effects on health, social life, and economics. Recently, vaccines from various companies have been urgently approved to control SARS-CoV-2 infections. However, any specific antiviral drug has not been confirmed so far for regular treatment. An important target is the main protease (M pro ), which plays a major role in replication of the virus. In this study, Gaussian and residue network models are employed to reveal two distinct potential allosteric sites on M pro that can be evaluated as drug targets besides the active site. Then, Food and Drug Administration (FDA)-approved drugs are docked to three distinct sites with flexible docking using AutoDock Vina to identify potential drug candidates. Fourteen best molecule hits for the active site of M pro are determined. Six of these also exhibit high docking scores for the potential allosteric regions. Full-atom molecular dynamics simulations with MM-GBSA method indicate that compounds docked to active and potential allosteric sites form stable interactions with high binding free energy (∆G bind ) values. ∆G bind values reach -52.06 kcal/mol for the active site, -51.08 kcal/mol for the potential allosteric site 1, and - 42.93 kcal/mol for the potential allosteric site 2. Energy decomposition calculations per residue elucidate key binding residues stabilizing the ligands that can further serve to design pharmacophores. This systematic and efficient computational analysis successfully determines ivermectine, diosmin, and selinexor currently subjected to clinical trials, and further proposes bromocriptine, elbasvir as M pro inhibitor candidates to be evaluated against SARS-CoV-2 infections., (© 2021 Wiley Periodicals LLC.)

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 pyrethroid deltamethrin and the macrocyclic lactone emamectin benzoate (EMB) are used to treat infestations of farmed salmon by parasitic salmon lice, Lepeophtheirus salmonis. While the efficacy of both compounds against Atlantic populations of the parasite has decreased as a result of the evolution of resistance, the molecular mechanisms of drug resistance in L. salmonis are currently not fully understood. The functionally diverse carboxylesterases (CaE) family includes members involved in pesticide resistance phenotypes of terrestrial arthropods. The present study had the objective to characterize the CaE family in L. salmonis and assess its role in drug resistance. L. salmonis CaE homologues were identified by homology searches in the parasite's transcriptome and genome. The transcript expression of CaEs predicted to be catalytically competent was studied using quantitative reverse-transcription PCR in drug susceptible and multi-resistant L. salmonis. The above strategy led to the identification of 21 CaEs genes/pseudogenes. Phylogenetic analyses assigned 13 CaEs to clades involved in neurodevelopmental signaling and cell adhesion, while three sequences were predicted to encode secreted enzymes. Ten CaEs were identified as being potentially catalytically competent. Transcript expression of acetylcholinesterase (ace1b) was significantly increased in multi-resistant lice compared to drug-susceptible L. salmonis, with transcript abundance further increased in preadult-II females following EMB exposure. In summary, results from the present study demonstrate that L. salmonis possesses fewer CaE gene family members than most arthropods characterized so far. Drug resistance in L. salmonis was associated with overexpression of ace1b., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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 increasing world-wide demand for food has prompted the development of efficient and environmentally friendly pesticide formulations. In this article, we have prepared CMC-g-PRSG carrier based on two compounds from natural materials carboxymethyl cellulose (CMC) and rosin (RS). The model pesticide avermectin (AVM) was encapsulated through hydrophobic interaction, and self-assembled to form nanopesticide AVM@CMC-g-PRSG with an average particle size of 167 nm. The prepared nanopesticide displays enhanced dispersibility and stability of AVM in water, and can effectively adhere to the leaves to prevent loss. The release rate of AVM encapsulated in the nanocarrier can be controlled by adjusting pH, and AVM half-life under ultraviolet radiation shows a 3-fold increase allowing control of pests for prolonged periods of time in practical applications. Biological safety tests showed that AVM@CMC-g-PRSG effectively reduces the toxicity of AVM to aquatic animals. Therefore, the cheap and degradable carrier CMC-g-PRSG can improve the effect of hydrophobic pesticides., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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.

We investigate the effect of the alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. Membrane incorporation of the purified CYP2E1 into HLM considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. It also eliminates the activating effect of α-naphthoflavone (ANF) seen in some HLM samples. To probe the physiological relevance of these effects, we compared three pooled preparations of HLM from normal donors (HLM-N) with a pooled preparation from ten heavy alcohol consumers (HLM-A). The composition of the P450 pool in all samples was characterized by the mass-spectrometric determination of 11 cytochrome P450 species. The fractional content of CYP2E1 in HLM-A was from 2.0 to 3.4 times higher than in HLM-N. In contrast, the content of CYP3A4 in HLM-A was the lowest among all samples. Despite that, HLM-A exhibited a much higher metabolism rate and a lower homotropic cooperativity with BQ, similar to CYP2E1-enriched HLM-N. To substantiate the involvement of interactions between CYP2E1 and CYP3A4 in these effects, we probed hetero-association of these proteins in CYP3A4-containing Supersomes™ with a technique employing CYP2E1 labeled with BODIPY-618 maleimide. These experiments evinced the interactions between the two enzymes and revealed an inhibitory effect of ANF on their association. Our results demonstrate that the functional properties of CYP3A4 are fundamentally dependent on the composition of the cytochrome P450 ensemble and suggest a possible impact of chronic alcohol exposure on the pharmacokinetics of drugs metabolized by CYP3A4., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Pentameric ligand-gated ion channels (pLGICs) are receptor proteins that are sensitive to their membrane environment, but the mechanism for how lipids modulate function under physiological conditions in a state dependent manner is not known. The glycine receptor is a pLGIC whose structure has been resolved in different functional states. Using a realistic model of a neuronal membrane coupled with coarse-grained molecular dynamics simulations, we demonstrate that some key lipid-protein interactions are dependent on the receptor state, suggesting that lipids may regulate the receptor's conformational dynamics. Comparison with existing structural data confirms known lipid binding sites, but we also predict further protein-lipid interactions including a site at the communication interface between the extracellular and transmembrane domain. Moreover, in the active state, cholesterol can bind to the binding site of the positive allosteric modulator ivermectin. These protein-lipid interaction sites could in future be exploited for the rational design of lipid-like allosteric drugs., Competing Interests: The authors have declared that no competing interests exist.

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.)

Background: Parascaris univalens is a pathogenic parasite of foals and yearlings worldwide. In recent years, Parascaris spp. worms have developed resistance to several of the commonly used anthelmintics, though currently the mechanisms behind this development are unknown. The aim of this study was to investigate the transcriptional responses in adult P. univalens worms after in vitro exposure to different concentrations of three anthelmintic drugs, focusing on drug targets and drug metabolising pathways., Methods: Adult worms were collected from the intestines of two foals at slaughter. The foals were naturally infected and had never been treated with anthelmintics. Worms were incubated in cell culture media containing different concentrations of either ivermectin (10 -9  M, 10 -11  M, 10 -13  M), pyrantel citrate (10 -6  M, 10 -8  M, 10 -10  M), thiabendazole (10 -5  M, 10 -7  M, 10 -9  M) or without anthelmintics (control) at 37 °C for 24 h. After incubation, the viability of the worms was assessed and RNA extracted from the anterior region of 36 worms and sequenced on an Illumina NovaSeq 6000 system., Results: All worms were alive at the end of the incubation but showed varying degrees of viability depending on the drug and concentration used. Differential expression (Padj < 0.05 and log2 fold change ≥ 1 or ≤ - 1) analysis showed similarities and differences in the transcriptional response after exposure to the different drug classes. Candidate genes upregulated or downregulated in drug exposed worms include members of the phase I metabolic pathway short-chain dehydrogenase/reductase superfamily (SDR), flavin containing monooxygenase superfamily (FMO) and cytochrome P450-family (CYP), as well as members of the membrane transporters major facilitator superfamily (MFS) and solute carrier superfamily (SLC). Generally, different targets of the anthelmintics used were found to be upregulated and downregulated in an unspecific pattern after drug exposure, apart from the GABA receptor subunit lgc-37, which was upregulated only in worms exposed to 10 -9  M of ivermectin., Conclusions: To our knowledge, this is the first time the expression of lgc-37 and members of the FMO, SDR, MFS and SLC superfamilies have been described in P. univalens and future work should be focused on characterising these candidate genes to further explore their potential involvement in drug metabolism and anthelmintic resistance.

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.

Glutathione S-transferases (GSTs) are a multifunctional protein superfamily that can catalyze the detoxification processes in an organism. In the present study, we determined the structure and function of GSTs in Chinese mitten crab (Eriocheir sinensis) by gene cloning, expression, and enzyme activity in order to investigate the metabolic detoxification of GSTs in the hepatopancreas and muscles under three pesticide (trichlorfon, β-cypermethrin and avermectin) stresses. Multiple sequence alignment analysis showed that all the three Es-GST genes possessed N-terminal, and C-terminal domain as well as G-binding sites, while Es-GST2 and Es-GST3 contained Mu-type GST-specific Mu-loop structures. Phylogenetic tree analysis revealed that the three Es-GSTs belonged to the Mu-type GST of crustaceans. The quantitative real-time PCR revealed that the three Es-GSTS were expressed in 9 tissues of Eriocheir sinensis, with highest expression in hepatopancreas and muscle. The expression of the three Es-GSTS significantly increased in the hepatopancreas and muscle under the three pesticide stresses compared to the control group, and a steady increase in GST activity was observed. The study showed that the three Es-GSTs belong to the Mu-type GST of the crustaceans and might play an important role in the metabolic detoxification in Eriocheir sinensis., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

Genetic engineering is a powerful method to improve the fermentation yield of bacterial metabolites. Since many biosynthetic mechanisms of bacterial metabolites have been unveiled, genetic engineering approaches have been applied to various issues of biosynthetic pathways, such as transcription, translation, post-translational modification, enzymes, transporters, etc. In this article, natamycin, avermectins, gentamicins, piperidamycins, and β-valienamine have been chosen as examples to review recent progress in improving their production by genetic engineering approaches. In these cases, not only yields of target products have been increased, but also yields of by-products have been decreased, and new products have been created., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

BldD generally functions as a repressor controlling morphological development of Streptomyces. In this work, evidences that BldD also activates antibiotic production are provided. In Streptomyces roseosporus (which produces daptomycin widely used for treatment of human infections), deletion of bldD notably reduced daptomycin production, but enhanced sporulation. BldD stimulated daptomycin production by directly activating transcription of dpt structural genes and dptR3 (which encodes an indirect activator of daptomycin production), and repressed its own gene. BldD-binding sites on promoter regions of dptE, dptR3, and bldD were all found to contain BldD box-like sequences, facilitating prediction of new BldD targets. Two Streptomyces global regulatory genes, adpA and afsR, were confirmed to be directly activated by BldD. The protein AfsR was shown to act as an activator of daptomycin production, but a repressor of development. BldD directly represses nine key developmental genes. In Streptomyces avermitilis (which produces effective anthelmintic agents avermectins), BldD homolog (BldDsav) directly activates avermectin production through ave structural genes and cluster-situated activator gene aveR. This is the first report that BldD activates antibiotic biosynthesis both directly and via a cascade mechanism. BldD homologs are widely distributed among Streptomyces, our findings suggest that BldD may activate antibiotic production in other Streptomyces species., (© 2019 John Wiley & Sons Ltd.)

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.)

The extensive application of pesticides in agricultural activities has raised increasing concerns on crop contamination by pesticide residues. Vegetables seem more susceptible to pesticide contamination given the high-intensive application of pesticides during their entire growth, while information about transfer and cell diffusion characteristics of pesticides in vegetables is currently insufficient. Here, we investigated the uptake, translocation and subcellular distribution behaviors of four commonly used pesticides in Chinese cabbage (Brassica rapa var. chinensis) under laboratory hydroponic conditions. Root uptake of pesticides followed the order of fenbuconazole > avermectin > thiamethoxam > spirotetramat. Thiamethoxam was more readily to be translocated from vegetable root to shoot, while spirotetramat, fenbuconazole and avermectin preferentially accumulated in vegetable root. Cell soluble components were the dominant storage compartment for thiamethoxam. The majority of spirotetramat, fenbuconazole and avermectin were partitioned into the cell walls. Hopefully, results of this study would extend the current knowledge of pesticide bioconcentration behavior in food-crops and assist in properly evaluating the threats of pesticide residues to human health via food chain., (Copyright © 2019. Published by Elsevier Inc.)

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.

Spider mites are destructive arthropod pests on many crops and they have developed resistance to nearly all acaricides. In recent years, along with the application of high throughput sequencing, the molecular mechanisms of mite resistance had made a series of progress. But, the response in molecular level of mite exposure to acaricides, as well as the original mechanism of resistance development was still unclear. To disclose the deeply mechanisms, we used RNA sequencing to analyze the responses of mite exposure to a sublethal concentration (LC 30 ) treatment of the three different action mode acaricides (Abamectin, Fenpropathrin, and Tebufenpyrad). A high number of differentially expressed genes may well be involved in detoxification and regulatory, with extensive overlap in differentially expressed genes between the three insecticide treatments. Two cytochrome P450 genes were co-up-regulated and one glutathione S-transferase genes were co-down-regulated in all the treatments, while carboxylesterase genes only had a response to abamectin. This interesting phenomenon revealed that P450 enzymes play an important role in the early stage of mite exposure to acaricide. Moreover, a P8 nuclear receptor gene was in response to stress caused by exposure to acaricides and RNA interference (RNAi) experiment indicated P8 nuclear receptor regulates the P450 enzyme activity and susceptibility of mites to acaricide. The differential response information of gene expression based on a large-scale sequence would provide some useful clues for studying the molecular mechanisms of mite resistance formation and development., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.