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The X-ray crystal structure of the epoxide hydrolase Lsd19 in complex with its substrate and product analogue is determined, providing insight into a general mechanism of enzyme-catalysed formation of polyether natural products. How enzymes achieve an unfavourable cyclization Polyethers are among the most complex chemical structures found in nature, and include products with anticancer, antibiotic and neuroprotective activity. The precise mechanism by which natural enzymes achieve the disfavoured cyclizations required in synthesis of such products remains obscure, but now Chu-Young Kim and colleagues characterize an epoxide hydrolase from a soil bacterium that catalyses the epoxide-opening cyclization of bisepoxyprelasalocid A to form the polyether lasalocid A. The reaction violates rules set out by Jack Baldwin in 1976 that are still used as guidelines for designing cyclization reactions. On the basis of structural and computational studies, the authors propose a general mechanism for the enzyme-catalysed formation of polyether natural products. Polycyclic polyether natural products have fascinated chemists and biologists alike owing to their useful biological activity, highly complex structure and intriguing biosynthetic mechanisms. Following the original proposal for the polyepoxide origin of lasalocid and isolasalocid 1 and the experimental determination of the origins of the oxygen and carbon atoms of both lasalocid and monensin, a unified stereochemical model for the biosynthesis of polyether ionophore antibiotics was proposed 2 . The model was based on a cascade of nucleophilic ring closures of postulated polyepoxide substrates generated by stereospecific oxidation of all- trans polyene polyketide intermediates 2 . Shortly thereafter, a related model was proposed for the biogenesis of marine ladder toxins, involving a series of nominally disfavoured anti-Baldwin, endo -tet epoxide-ring-opening reactions 3 , 4 , 5 . Recently, we identified Lsd19 from the Streptomyces lasaliensis gene cluster as the epoxide hydrolase responsible for the epoxide-opening cyclization of bisepoxyprelasalocid A 6 to form lasalocid A 7 , 8 . Here we report the X-ray crystal structure of Lsd19 in complex with its substrate and product analogue 9 to provide the first atomic structure—to our knowledge—of a natural enzyme capable of catalysing the disfavoured epoxide-opening cyclic ether formation. On the basis of our structural and computational studies, we propose a general mechanism for the enzymatic catalysis of polyether natural product biosynthesis.

Polyether ionophores are complex natural products capable of transporting cations across biological membranes. Many polyether ionophores possess potent antimicrobial activity and a few selected compounds have the ability to target aggressive cancer cells. Nevertheless, ionophore function is believed to be associated with idiosyncratic cellular toxicity and, consequently, human clinical development has not been pursued. Here, we demonstrate that structurally novel polyether ionophores can be efficiently constructed by recycling components of highly abundant polyethers to afford analogues with enhanced antibacterial selectivity compared to a panel of natural polyether ionophores. We used classic degradation reactions of the natural polyethers lasalocid and monensin and combined the resulting fragments with building blocks provided by total synthesis, including halogen-functionalized tetronic acids as cation-binding groups. Our results suggest that structural optimization of polyether ionophores is possible and that this area represents a potential opportunity for future methodological innovation.Polyether ionophores are natural products that display antibacterial activity—but they also show activity against mammalian cells, which has limited their development as clinical antibiotics. Now, a semisynthesis principle of recycling substructures from highly abundant natural polyether ionophores has been used to prepare analogues with enhanced selectivity towards bacterial cells.

Salinomycin (SAL) and lasalocid (LAS) are widely used as ionophore antibiotics for coccidiosis control. However, their common use as feed additives has led to the occurrence of feed cross-contamination, which has toxic effects on non-target animals. There have been few reports on multiple-residue detection for SAL and LAS in recent years. In this study, two single-chain antibody fragments (scFvs) capable of specifically recognizing SAL and LAS were constructed. Using LAS-scFv and SAL-scFv as parent antibodies, a complete bispecific single-chain diabody (scDb) against both LAS and SAL was built using splicing by overlap extension polymerase chain reaction (SOE-PCR). In addition, the key amino acid sites and interaction energy of antibody variable regions for small-molecule recognition were preliminarily studied by homology modeling and molecular docking. Finally, IC50 values of 12.9 and 8.6 ng/mL, with a linear range of 6.9–24.0 and 4.7–16.0 ng/mL, were obtained for LAS-scFv and SAL-scFv, respectively. An indirect competitive enzyme-linked immunosorbent assay (icELISA) method was established using scDb to obtain an IC50 of 3.5 ng/mL for LAS and 4.1 ng/mL for SAL, which showed better sensitivity and specificity than those of the parent scFv antibodies. The recoveries of LAS and SAL in chicken liver were 89.2–92.7%(CV<4.7%) and 88.6–90.2% (CV<6.8%)), respectively.

Enzymes used in the synthesis of natural products are potent catalysts, capable of efficient and stereoselective chemical transformations. Lsd18 catalyzes two sequential epoxidations during the biosynthesis of lasalocid A, a polyether polyketide natural product. We performed protein engineering on Lsd18 to improve its thermostability and catalytic activity. Utilizing structure-guided methods of FoldX and Rosetta-ddG, we designed 15 mutants of Lsd18. Screening of these mutants using thermal shift assay identified stabilized variants Lsd18-T189M, Lsd18-S195M, and the double mutant Lsd18-T189M-S195M. Trypsin digestion, molecular dynamic simulation, circular dichroism (CD) spectroscopy, and X-ray crystallography provided insights into the molecular basis for the improved enzyme properties. Notably, enhanced hydrophobic interaction within the enzyme core and interaction of the protein with the FAD cofactor appear to be responsible for its better thermostability.

Carboxylic ionophores are polyether antibiotics used in production animals as feed additives, with a wide range of benefits. However, ionophore toxicosis often occurs as a result of food mixing errors or extra-label use and primarily targets the cardiac and skeletal muscles of livestock. The ultrastructural changes induced by 48 hours of exposure to 0.1 μM monensin, salinomycin, and lasalocid in cardiac (H9c2) and skeletal (L6) myoblasts in vitro were investigated using transmission electron microscopy and scanning electron microscopy. Ionophore exposure resulted in condensed mitochondria, dilated Golgi apparatus, and cytoplasmic vacuolization which appeared as indentations on the myoblast surface. Ultrastructurally, it appears that both apoptotic and necrotic myoblasts were present after exposure to the ionophores. Apoptotic myoblasts contained condensed chromatin and apoptotic bodies budding from their surface. Necrotic myoblasts had disrupted plasma membranes and damaged cytoplasmic organelles. Of the three ionophores, monensin induced the most alterations in myoblasts of both cell lines.

Use of ionophores in cattle diets has been proposed as a strategy for mitigation of enteric CH₄ emissions. Short- and long-term effects of feeding a single ionophore (monensin) or rotation of 2 ionophores (monensin and lasalocid) on enteric CH₄ emissions were evaluated in 36 Angus yearling steers (328 ± 24.9 kg of BW) over a 16-wk period. Steers were randomly assigned to 6 dietary treatments of 6 steers each. The 6 diets were low-concentrate without ionophore supplementation, low-concentrate with monensin supplementation, low-concentrate with a 2-wk rotation of monensin and lasalocid supplementation, high-concentrate without ionophore supplementation, high-concentrate with monensin supplementation, and high-concentrate with a 2-wk rotation of monensin and lasalocid supplementation. Daily enteric CH₄ emissions, as measured using the SF₆ tracer gas technique, ranged from 54.7 to 369.3 L/steer daily. Supplementing ionophores decreased (P < 0.05) enteric CH₄ emissions, expressed as liters per kilogram of DMI or percentage of GE intake, by 30% for the first 2 wk and by 27% for the first 4 wk, for cattle receiving the high-concentrate and low-concentrate diets, respectively. Cattle fed a rotation of ionophores did not (P > 0.05) exhibit a greater decrease and did not (P > 0.05) have a longer period of depressed enteric CH₄ emissions compared with cattle receiving monensin only. Ionophore supplementation did not (P > 0.05) alter total ruminal fluid VFA concentration; however, the acetate:propionate ratio and ammonia-N concentration in ruminal fluid were decreased (P < 0.001) from the time that ionophores were introduced to the time they were removed from the diets. Both monensin and the rotation of monensin and lasalocid decreased (P < 0.001) total ciliate protozoal populations by 82.5% in the first 2 wk and by 76.8% in the first 4 wk during which they were supplemented in the high-concentrate and low-concentrate diets, respectively. Original ciliate protozoal populations were restored by the fourth and sixth week of supplementation when cattle were fed the high- or low-concentrate diets, respectively. No significant change was observed thereafter. These data suggest that the effects of ionophores on enteric CH₄ production are related to ciliate protozoal populations and that ciliate protozoal populations can adapt to the ionophores present in either low- or high-concentrate diets. Rotation of monensin and lasalocid did not (P > 0.05) prevent ciliate protozoal adaptation to ionophores.

Monensin and lasalocid are polyether ionophores commonly used in the beef and poultry industries for the prevention of coccidial infections and promotion of growth. These ionophores can exhibit higher toxicity than many other antibiotics; thus, evaluating their fate in the environments associated with concentrated feed operations is important. Sorption of monensin and lasalocid was measured in eight soils of varying physiochemical composition. Organic carbon-normalized sorption coefficients (log K(OC)) ranged from 2.1 to 3.8 for monensin and from 2.9 to 4.2 for lasalocid and were inversely correlated to equilibrium soil-solution pH. Degradation of lasalocid and monensin in two contrasting soils with and without manure amendment was measured in moist soils at 23°C and 0.03 MPa moisture potential. The half-life of both compounds in the fresh nonsterile soils was less than 4 d, for which monensin degraded slightly faster than lasalocid. Fresh liquid manure amendments did not significantly alter degradation of either compound. Based on parallel 60Co-sterilized soil experiments, some abiotic degradation of monensin was apparent, whereas lasalocid only degraded in the presence of microbes. Analysis of beef-derived lagoon effluent used for irrigation confirmed that monensin can be present at low-ppb to low-ppm concentrations in the aqueous and suspended solids fractions, respectively; however, subsequent analysis of drainage water in a nearby ditch suggested that attenuation by soil after land application will greatly reduce the amount entering surface waters.

Mastitis is a major disease of dairy cattle. Given the recent emergence of methicillin-resistant Staphylococcus aureus as a cause of bovine mastitis, new intramammary (IMA) treatments are urgently required. Lasalocid, a member of the polyether ionophore class of antimicrobial agents, has not been previously administered to cows by the IMA route and has favorable characteristics for development as a mastitis treatment. This study aimed to develop an IMA drug delivery system (IMDS) of lasalocid for the treatment of bovine mastitis. Minimum inhibitory concentrations (MICs) were determined applying the procedures recommended by the Clinical and Laboratory Standards Institute. Solid dispersions (SDs) of lasalocid were prepared and characterized using differential scanning calorimetry and Fourier transform infrared spectroscopy. IMDSs containing lasalocid of micronized, nano-sized, or as SD form were tested for their IMA safety in cows. Therapeutic efficacy of lasalocid IMDSs was tested in a bovine model involving experimental IMA challenge with the mastitis pathogen Streptococcus uberis. Lasalocid demonstrated antimicrobial activity against the major Gram-positive mastitis pathogens including S. aureus (MIC range 0.5-8 μg/mL). The solubility test confirmed limited, ion-strength-dependent water solubility of lasalocid. A kinetic solubility study showed that SDs effectively enhanced water solubility of lasalocid (21-35-fold). Polyvinylpyrrolidone (PVP)-lasalocid SD caused minimum mammary irritation in treated cows and exhibited faster distribution in milk than either nano or microsized lasalocid. IMDSs with PVP-lasalocid SD provided effective treatment with a higher mastitis clinical and microbiological cure rate (66.7%) compared to cloxacillin (62.5%). Lasalocid SD IMDS provided high cure rates and effectiveness in treating bovine mastitis with acceptable safety in treated cows.

A method for the simultaneous determination of robenidine, halofuginone, lasalocid, monensin, nigericin, salinomycin, narasin, and maduramicin residues in eggs by liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed. The sample preparation method used a combination of liquid–liquid extraction (LLE) and solid-phase extraction (SPE) technology to extract and purify these target compounds from eggs. The target compounds were separated by gradient elution using high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC). Tandem mass spectrometry was used to quantitatively and qualitatively analyze the target compounds via electrospray ionization (ESI+) and multiple reaction monitoring mode. The HPLC–MS/MS and UPLC–MS/MS methods were validated according to the requirements defined by the European Union and the Food and Drug Administration. The limits of detection and limits of quantification of the eight coccidiostats in eggs were 0.23–0.52 µg/kg and 0.82–1.73 µg/kg for HPLC–MS/MS, and 0.16-0.42 µg/kg and 0.81-1.25 µg/kg for UPLC–MS/MS, respectively. The eggs were spiked with four concentrations of the eight coccidiostats, and the HPLC–MS/MS and UPLC–MS/MS average recoveries were all higher than 71.69% and 72.26%, respectively. Compared with the HPLC–MS/MS method, utilizing UPLC–MS/MS had the advantages of low reagent consumption, a short detection time, and high recovery and precision. Finally, the HPLC–MS/MS and UPLC–MS/MS methods were successfully applied to detect eight coccidiostats in 40 eggs.

To assess the impact of monensin, lasalocid, and a 1:1 (w/w) mixture of monensin and lasalocid on the formation of Microcystis aeruginosa-based harmful algal blooms (HABs), laboratory experiments were conducted [temperature 20 ± 1°C, illumination PAR 30 ± 4 μmol/m2 ·s (12-h light/dark cycle), growth medium BG-11] at concentrations of 100, 200, 500, 1,000, and 2,000 μg/L. Measurements included optical density, cell protein, chlorophyll ‘a’ (Chl. a) content, oxidative stress, catalase activity (CAT), and guaiacol peroxidase activity (GPX). Monensin treatment showed a dose-dependent positive effect on M. aeruginosa growth at concentrations ≤500 μg/L and a gradual dose-dependent growth inhibition at concentrations of 1,000 and 2,000 μg/L. The results indicated that monensin has a significant positive effect on the formation of HABs by M. aeruginosa. Lasalocid treatment showed a growth reduction of M. aeruginosa at concentrations ≤500 μg/L and a growth increase at higher concentrations (1,000 and 2,000 μg/L). The 1:1 (w/w) mixture test showed an intermediate response, as indicated by the individual treatments signifying the potential interactive effects of these antibiotics on M. aeruginosa. Furthermore, alterations in Chl. ‘a’, oxidative stress, CAT, and GPX measurements provided evidence of the impact of these antibiotics on the existence of M. aeruginosa.