Combination of monensin and erlotinib synergistically inhibited the growth and cancer stem cell properties of triple-negative breast cancer by simultaneously inhibiting EGFR and PI3K signaling pathways.

BACKGROUND: Cancer stem cells (CSCs) in triple-negative breast cancer (TNBC) are recognized as a highly challenging subset of cells, renowned for their heightened propensity for relapse and unfavorable prognosis. Monensin, an ionophoric antibiotic, has been reported to exhibit significant therapeutic efficacy against various cancers, especially CSCs. Erlotinib is classified as one of the EGFR-TKIs and has been previously identified as a promising therapeutic target for TNBC. Our research aims to assess the effectiveness of combination of monensin and erlotinib as a potential treatment strategy for TNBC. METHODS: The combination of monensin and erlotinib was assessed for its potential anticancer activity through various in vitro assays, including cytotoxicity assay, colony formation assay, wound healing assay, transwell assay, mammosphere formation assay, and proportion of CSCs assay. Additionally, an in vivo study using tumor-bearing nude mice was conducted to evaluate the inhibitory effect of the monensin and erlotinib combination on tumor growth. RESULTS: The results indicated that combination of monensin with erlotinib synergistically inhibited cell proliferation, the migration rate, the invasion ability and decreased the CSCs proportion, and CSC markers SOX2 and CD133 in vivo and in vitro. Furthermore, the primary proteins involved in the signaling pathways of the EGFR/ERK and PI3K/AKT are simultaneously inhibited by the combination treatment of monensin and erlotinib in vivo and in vitro. CONCLUSIONS: The simultaneous inhibition of the EGFR/ERK and PI3K/AKT/mTOR signaling pathways by the combination of monensin and erlotinib exhibited a synergistic effect on suppressing tumor proliferation and cancer cell stemness in TNBC.

Gastrointestinal response to the early administration of antimicrobial agents in growing turkeys infected with Escherichia coli.

This study investigated the effects of the early administration of enrofloxacin (E) or doxycycline (D) for the first 5 consecutive days of life, or the continuous administration of the coccidiostat monensin (M) throughout the rearing period on gastrointestinal function in turkeys infected with avian pathogenic Escherichia coli (APEC) in an early or later stage of rearing. Experiment 1 lasted 21 d, and turkeys in groups E, D, and M were infected with APEC on d 15. Experiment 2 lasted 56 d, and it had a factorial arrangement of treatments where birds in groups E, D, and M were infected with APEC on d 15 or d 50. In both experiments, control groups (C) consisted of infected and uninfected birds without antibiotic or coccidiostat administration. On d 21 (Experiment 1) and d 56 (Experiment 2), 8 birds from each subgroup were killed, and the ileal and cecal digesta were sampled to analyze the activity of bacterial enzymes and the concentrations of short-chain fatty acids (SCFA). The experimental treatments did not affect the final body weight or body weight gain of birds. Both experiments demonstrated that APEC contributed to an increase in ammonia levels of the cecal digesta (means from 2 experiments: 0.311 vs. 0.225 mg/g in uninfected birds) and ileal pH (6.79 vs. 6.00) and viscosity (2.43 vs. 1.83 mPa⋅s). Moreover, the E. coli challenge enhanced the extracellular activity of several cecal bacterial enzymes, especially in older turkeys infected with APEC in a later stage of life. The continuous administration of monensin throughout the rearing period resulted in a weaker gastrointestinal response in older birds, compared with the other 2 antibiotics administered for the first 5 d of life. The results of the study are inconclusive as both desirable and undesirable effects of preventive early short-term antibiotic therapy were observed in turkeys, including normalization of ileal viscosity and cecal ammonia concentration (positive effect), and disruption in cecal SCFA production (negative effect).

A multi-approach analysis of the toxicity of a commercial formulation of monensin on Rhinella arenarum embryos and larvae.

Monensin, an antibacterial commonly used in animal fattening, can enter aquatic ecosystems and harm non-target organisms. Since there are no previous studies about the effects of monensin on amphibians, the aim of the present study was to evaluate the lethal and sublethal toxicity of a commercial formulation of monensin (CFM) through standardized bioassays with embryos and larvae of the amphibian Rhinella arenarum. Oxidative stress (catalase and glutathione S-transferase activities, and reduced glutathione and lipid peroxidation levels), cholinesterasic effect (acetylcholinesterase and butyrylcholinesterase activities) and mutagenicity (micronuclei frequency) biomarkers were evaluated. The CFM produced teratogenic effects, with a teratogenic index of 6.21. Embryos (504 h-LC50: 273.33 µg/L) were more sensitive than larvae, as no significant mortality was observed on larvae exposed up to 3000 µg/L for 504 h. However, oxidative stress, cholinesterasic effect and mutagenicity biomarkers were altered on larvae exposed for 96 h to environmentally relevant concentrations (4, 12 and 20 µg/L of monensin active ingredient). The CFM caused adverse effects on the exposed organisms, primarily on embryos, leading to lethal and sublethal effects, which could impact the wildlife when it reaches aquatic ecosystems.

Influence of low-level tannin supplementation on comparative growth performance of Holstein and Angus × Holstein cross calf-fed concentrate-based finishing diets for 328 d.

The objective of the current study was to evaluate the effects of tannin and monensin supplementation in feedlot diets and breed (Holstein vs. Angus × Holstein) on growth performance, energetic efficiency, and carcass characteristics. Eighty purebred Holstein calves (HOL; initial body weight (BW) = 130 ±â€…5 kg) and 80 Angus × Holstein calves (AXH; initial BW = 129 ±â€…6 kg) were blocked by initial BW and randomly assigned to 40 pens. Dietary treatments consisted of a steam-flaked corn-based diet supplemented with (1) no feed additive (CON); (2) 30 mg of monensin/kg of dry matter (DM; MON; Rumensin 90, Elanco, Greenfield, IN); (3) 1.5 g tannin)/kg of DM (TAN; ByPro, 70% condensed tannin, SilvaFeed, Indunor, S.A., Buenos Aires, Argentina); (4) M + T, the combination of MON plus TAN dietary treatments. Data were analyzed as a randomized complete block in a 2 × 4 factorial arrangement of treatments, using pens as experimental units. There were no interactions (P > 0.05) between feed additives and breed. Supplemental MON increased (P ≤ 0.04) initial 112-d BW and gain efficiency. However, there were no dietary treatment effects (P > 0.10) on overall growth performance. Monensin supplementation decreased (P = 0.04) minimum daily ruminal temperature compared with other dietary treatments during July, but TAN did not affect ruminal temperature. Holstein steers had greater (P = 0.04) overall DM intake compared with AXH, with no difference (P = 0.19) in overall ADG, leading to increased (P < 0.01) gain efficiency for AXH compared with HOL. Dietary net energy for maintenance and gain, based on growth performance, were greater (P ≤ 0.01) for AXH vs HOL. Compared with HOL, AXH steers had greater (P ≤ 0.01) carcass weight, dressing percentage, kidney, pelvic, and heart fat, 12th rib fat thickness, longissimus area, and preliminary yield grade. Holstein steers had lower (P ≤ 0.04) minimum average ruminal temperature during June compared with AXH, with no differences (P ≥ 0.14) between breeds during July or August. Results indicate that feed additives did not appreciably affect steer growth performance and carcass characteristics, but crossbred AXH steers had greater growth performance, efficiency of dietary energy utilization, and carcass quality measures compared with HOL. This study observed a reduction (4.7%) in maintenance energy expenditure in AXH compared with HOL, implying in maintenance energy coefficient of 0.086 vs 0.082 for HOL and AXH, respectively.

Effects of tannin and monensin supplementation on growth performance, energetic efficiency, and carcass characteristics were evaluated in Holstein and Angus × Holstein steers. The investigation used a factorial design to access the impacts of both feed additives and breed on the study's parameters. Tannin supplementation did not affect growth performance. There were no dietary treatment effects on overall steer growth performance. Calf Holstein steers were fed with grain diet based. Holstein steers had greater overall DM intake than Angus × Holstein steers, but breed did not affect average daily gain. Thus, gain efficiency was greater for Angus × Holstein vs Holstein steers. There was no effect of dietary treatment on carcass measures. Compared with Holsteins, Angus × Holstein steers had greater carcass weight, dressing percentage, internal and external fat, longissimus area, and marbling score than Holstein steers. The current study suggests that monensin and tannin supplementation did not affect overall steer growth performance and carcass characteristics. Compared with Holsteins, crossbred Angus × Holstein steers had increased growth performance and carcass quality measures.

Effect of monensin and live-cell yeast supplementation on lactation performance, feeding behavior, and total-tract nutrient digestibility in dairy cows.

The objective of this study was to evaluate the effects of supplementing monensin (19.8 g/Mg DM TMR; MON) and Saccharomyces cerevisiae CNCM I-1077 live-cell yeasts (Saccharomyces cerevisiae CNCM I-1077; 1 × 1010 cfu/head per day; LCY) on lactation performance, feeding behavior, and total-tract nutrient digestibility of high-producing dairy cows. Sixty-four multiparous Holstein cows (3.2 ± 1.5 lactations; 97 ± 16 DIM, and 724 ± 68 kg of BW at covariate period initiation) and 32 gate feeders were enrolled in a study with a completely randomized design and a 2 × 2 factorial arrangement. Cows and gate feeders were randomly assigned to treatments (16 cows and 8 gate feeders per treatment). Cows were allowed 2 wk to acclimate to feeding gates followed by a 2-wk covariate period. During the acclimation and covariate periods, all cows were fed a diet containing MON and LCY. Following the covariate period, cows were enrolled in a 10-wk treatment period during which cows were randomly assigned to 1 of 4 treatments: (1) a combination of MON and LCY (MON-LCY), (2) MON-CON, (3) CON-LCY, or (4) neither MON nor LCY (CON-CON). Data were analyzed using a mixed model with week as a repeated measure and fixed effects of MON, LCY, week, and all their interactions. Cow (treatment) was included as a random effect. The average covariate period value of each variable was used as a covariate. Three-way interactions were observed for DMI and feed efficiency. Dry matter intake decreased from wk 4 to 5 and wk 8 to 10 in MON-LCY cows compared with CON-CON. No treatment differences were observed for actual or component-corrected milk yield or milk components, except for a tendency for LCY to decrease milk fat yield. Feed efficiency was greater for MON-LCY relative to CON-CON in 4 of 10 wk. Interactions between MON and LCY were observed for dry matter and organic matter digestibility, where both were lower for CON-CON than other treatments. Under the conditions of the present study, feeding dairy cows in a high feed bunk density a combination of MON and LCY can decrease intake and improve feed efficiency without affecting milk production or components. Additionally, monensin and live-cell yeasts may each improve total-tract digestibility based on improvements in DM and OM digestibility.

Comparison of solubility profiles for pioneer and generic monensin premixes in biorelevant simulated intestinal fluid based on shake flask extractions.

In the United States, a generic Type A medicated article product can gain the FDA approval by demonstrating bioequivalence (BE) to the pioneer product by successfully conducting a blood level, pharmacodynamic, or clinical BE study. A biowaiver can be granted based on several criteria, assuming the dissolution of the test and reference products represents the only factor influencing the relative bioavailability of both products. Monensin is practically insoluble in H2O per the USP definition. Previously published data from a comparison study of monensin dissolution profiles from the pioneer product and four generic products using biorelevant media showed that generic monensin products demonstrated different dissolution profiles to the pioneer product in these USP biorelevant rumen media. This follow-up study compared the solubility profiles in simulated intestinal fluid (cFaSSIF, pH 7.5) for the pioneer product and four generic products. The generic monensin products demonstrated different in vitro dissolution profiles to the pioneer product in biorelevant media. The differences demonstrated in solubility and dissolution profiles are of concern regarding the potential efficacy of generic monensin in cattle. There are also additional concerns for the potential development of Eimeria resistance in cattle receiving a sub-therapeutic dose of monensin from a less soluble generic product.

Simultaneous determination of selected ionophoric coccidiostats and amino acids in feed premixes using high-performance liquid chromatography-ultraviolet detection method.

The combination of ionophoric coccidiostats and amino acids (AAs) is important in poultry feeding to enhance immunity and improve the growth and feed efficiency of birds suffering from coccidiosis. A simple, rapid, and economical high-performance liquid chromatography-ultraviolet detection (HPLC-UV) method for the simultaneous determination of three ionophoric coccidiostats, namely salinomycin (SAL), maduramicin (MAD), and monensin (MON) in addition to three AAs; L-tryptophan (L-TRP), alpha-ketoleucin (KLEU), and L-valine (L-VAL) in feed premixes was developed and validated. Chromatographic separation was achieved in less than 12 min using a phenyl hexyl column with a mobile phase consisting of acetonitrile/methanol/water (25:20:55, v/v/v) adjusted to pH 3 using phosphoric acid. Isocratic elution was performed at a flow rate of 1 mL/min with UV detection at 210 nm. The method showed good linearity in the ranges 0.50-5.0 mg/mL for MON, 0.20-2.0 mg/mL for MAD and SAL, 10.0-100.0 µg/mL for L-TRP and KLEU, and 50.0-500.0 µg/mL for VAL. The developed method was successfully applied to determine the studied analytes in feed premixes with good recoveries and precision. The good validation criteria of the proposed method allow its utilization in quality control laboratories.

Effects of allicin on growth performance, antioxidant profile, and microbiota compared to monensin of growing goats.

Allicin is a sulfur-containing compound extracted from raw garlic (Allium sativum L.). We compared the effect of allicin addition on growth performance, serum biochemical parameters, and rumen microbiota of goats compared to monensin. Twenty-four Anhui white goats were assigned randomly to one of three dietary treatments: 1) a basal diet (CON); 2) the basal diet with allicin addition at 750 mg per head per day (AC); 3) the basal diet with monensin addition at 30 mg per kg of diet (MS). Animals were fed for 8 weeks. Results showed the average daily gain, and feed efficiency was increased with allicin and monensin addition. Serum levels of IgG, total superoxide dismutase, and glutathione peroxidase were higher in the AC group than those in the CON and MS groups. The microbiota analysis revealed that monensin addition mainly affected genera related to carbohydrate and protein metabolism, and allicin mainly affected genera related to energy metabolism and intestinal health. In conclusion, allicin could improve growth performance and have advantages over monensin in improving the antioxidant capacity and immune function of goats. Allicin may be a potential alternative to monensin.

Staphylococcus aureus mutants resistant to the feed-additive monensin show increased virulence and altered purine metabolism.

Ionophores are antibacterial compounds that affect bacterial growth by changing intracellular concentrations of the essential cations, sodium and potassium. They are extensively used in animal husbandry to increase productivity and reduce infectious diseases, but our understanding of the potential for and effects of resistance development to ionophores is poorly known. Thus, given their widespread global usage, it is important to determine the potential negative consequences of ionophore use on human and animal health. In this study, we demonstrate that exposure to the ionophore monensin can select for resistant mutants in the human and animal pathogen Staphylococcus aureus, with a majority of the resistant mutants showing increased growth rates in vitro and/or in mice. Whole-genome sequencing and proteomic analysis of the resistant mutants show that the resistance phenotype is associated with de-repression of de novo purine synthesis, which could be achieved through mutations in different transcriptional regulators including mutations in the gene purR, the repressor of the purine de novo synthesis pathway. This study shows that mutants with reduced susceptibility to the ionophore monensin can be readily selected and highlights an unexplored link between ionophore resistance, purine metabolism, and fitness in pathogenic bacteria.IMPORTANCEThis study demonstrates a novel link between ionophore resistance, purine metabolism, and virulence/fitness in the key human and animal pathogen Staphylococcus aureus. The results show that mutants with reduced susceptibility to the commonly used ionophore monensin can be readily selected and that the reduced susceptibility observed is associated with an increased expression of the de novo purine synthesis pathway. This study increases our understanding of the impact of the use of animal feed additives on both human and veterinary medicine.

Effects of monensin supplementation on rumen fermentation, methane emissions, nitrogen balance, and metabolic responses of dairy cows: A systematic review and dose-response meta-analysis.

To investigate the effects of supplemental monensin administration on the metabolic responses of dairy cows, a systematic review and dose-response meta-analysis were conducted. Initially, 604 studies were identified through comprehensive database searches, including Google Scholar, Scopus, Science Direct, and PubMed, using key words related to dairy cows, monensin, and metabolic outcomes. After a 2-stage screening process, 51 articles with a total of 60 experiments were selected for meta-analysis based on criteria such as study implementation date between 2001 and 2022, presence of a control group that did not receive monensin supplementation, reporting of at least 1 outcome variable, and presentation of means and corresponding errors. The meta-analysis used the 1-stage random-effects method, and sensitivity analyses were performed to assess the robustness of the results. The results showed that the administration of monensin at a dosage of 19 to 26 mg/kg was inversely related to methane emissions and that the administration of monensin at a dosage of 18 to 50 mg/kg resulted in a significant decrease in dry matter intake. Administration of monensin at doses of 13 to 28 and 15 to 24 mg/kg also resulted in a significant decrease in ruminal acetate proportion and an increase in propionate proportion, respectively, with no effects on ruminal butyrate, NH3, or pH levels. We found no effects on blood parameters or nitrogen retention, but a significant negative correlation was observed between monensin supplementation and fecal nitrogen excretion. Based on the analysis of all variables evaluated, the optimal dose range of monensin was estimated to be 19 to 24 mg/kg.

Monensin poisoning outbreak in free-ranging and captive birds.

Monensin poisoning is uncommon and has been rarely reported in birds. This work aimed to described clinical-pathological aspects of an outbreak of monensin poisoning in captive and free-ranging birds. Thirty-seven of 600 captive birds fed a diet containing 893.19 mg/kg of monensin died within 10 days (mortality 6.17%). There was no ionophore antibiotics on the feed label supplied to captive birds, which established an error in feed production. Necropsies were performed on twelve animals: Muscovy duck (Cairina moschata) (2/12), greater rhea (Rhea americana) (2/12), black-necked swan (Cygnus melancoryphus) (2/12), garganey (Anas querquedula) (1/12), ostrich (Struthio camelus) (1/12), and common pigeon (Columbus livia) (4/12). These four common pigeons were free-ranging birds and died after eating the same contaminated feed. Birds were mainly found dead, however in animals which clinical signs were observed (Columba livia, Rhea americana, Cairina moschata, Anas querquedula, and Struthio camelus), they included incoordination, inability to stand, and intense prostration, that ranged from 24 to 72 h until death. Grossly, five birds had focally extensive pale firm areas in the myocardium and two had in the skeletal muscles, one being concomitant lesions. Histologically, muscle necrosis and degeneration were observed in striated musculature (skeletal and/or heart) in all birds analyzed. Monensin poisoning outbreaks can affect free-ranging birds that are fed on external feeders, as well as captive birds, due to an error in the feed formulation.

Effect of monensin on milk production efficiency and milk composition in lactating dairy cows fed modern diets.

Since the US Food and Drug Administration's approval of monensin in 2004, significant nutritional advances have been made to increase feed efficiency and milk fat production. Recent evidence suggests monensin's adverse effect on milk fat percentage may be absent when diets are formulated to address known diet-induced milk fat depression risk factors. Thus, study objectives were to evaluate effects of monensin level on dry matter intake (DMI), milk production and composition, and efficiency of high-producing cows fed diets formulated to optimize milk fat. Ninety-six lactating Holstein cows (36 primiparous, 60 multiparous; 106 ± 17 d in milk [DIM]) were balanced by parity, DIM, and milk production and were randomly assigned to 1 of 12 pens with 8 cows per pen. All cows received 11 g/t monensin for 5 wk after which pens received 1 of 4 dietary treatments (n = 3) formulated to provide 0 (CON), 11 (R11), 14.5 (R14.5), or 18 (R18) g/t monensin for 9 wk. The basal diet was 54% forage, 27% NDF, 29% starch, and 2.3% rumen unsaturated fatty acid load. Pen was the experimental unit and data were analyzed using the Fit Model Procedure of JMP. Effects of treatment, time, and treatment × time interaction were included as fixed effects and pen as a random effect. Least squares means were determined and linear and quadratic contrasts were tested. Dry matter intake tended to decrease linearly with increasing monensin dose. Milk yield, fat percentage, and protein percentage and yield were unaffected by treatment while fat yield was quadratically increased. Milk de novo and mixed fatty acid (FA) yields (g/d) increased quadratically with monensin whereas preformed FA linearly decreased during the experimental period. Energy-corrected milk (ECM) was quadratically increased by monensin. Milk urea nitrogen concentrations increased linearly with increasing monensin dose. Monensin linearly increased feed efficiency (ECM/DMI, 3.5% fat-corrected milk/DMI, and solids-corrected milk/DMI). Body weight gain did not differ between treatments. Estimated dietary energy tended to increase linearly with increasing monensin level. These data suggest monensin improves component-corrected milk production efficiency, estimated dietary energy, and does not negatively affect milk fat percentage or FA profile.

Effects of cashew nut shell extract and monensin on in vitro ruminal fermentation, methane production, and ruminal bacterial community.

The objective of this study was to evaluate the effects of cashew nut shell extract (CNSE) and monensin on ruminal in vitro fermentation, CH4 production, and ruminal bacterial community structure. Treatments were as follows: control (CON, basal diet without additives); 2.5 µM monensin (MON); 0.1 mg CNSE granule/g DM (CNSE100); and 0.2 mg CNSE granule/g DM (CNSE200). Each treatment was incubated with 52 mL of buffered ruminal content and 500 mg of total mixed ration for 24 h using serum vials. The experiment was performed as a complete randomized block design with 3 runs. Run was used as a blocking factor. Each treatment had 5 replicates, in which 2 were used to determine nutrient degradability, and 3 were used to determine pH, NH3-N, volatile fatty acids, lactate, total gas, CH4 production, and bacterial community composition. Treatment responses for all data, excluding bacterial abundance, were analyzed with the GLIMMIX procedure of SAS v9.4. Treatment responses for bacterial community structure were analyzed with a PERMANOVA test run with the R package vegan. Orthogonal contrasts were used to test the effects of (1) additive inclusion (ADD: CON vs. MON, CNSE100, and CNSE200); (2) additive type (MCN: MON vs. CNSE100 and CNSE200); and (3) CNSE dose (DOS: CNSE100 vs. CNSE200). We observed that pH, acetate, and acetate:propionate ratio in the CNSE100 treatment were lower compared with CNSE200, and propionate in the CNSE100 treatment was greater compared with CNSE200. Compared with MON, CNSE treatments tended to decrease total lactate concentration. Total gas production of CON was greater by 2.63% compared with all treatments, and total CH4 production was reduced by 10.64% in both CNSE treatments compared with MON. Also, compared with MON, in vitro dry matter degradabilities in CNSE treatments were lower. No effects were observed for NH3-N or in vitro neutral detergent fiber degradability. Finally, the relative abundances of Prevotella, Treponema, and Schwartzia were lower, whereas the relative abundances of Butyrivibrio and Succinivibrio were greater in all treatments compared with CON. Overall, the inclusion of CNSE decreased CH4 production compared with MON, making CNSE a possible CH4 mitigation additive in dairy cattle diets.

Effects of monensin sodium and live attenuated oocyst vaccine as coccidiosis management programs on productive performance, bone quality and mineral utilisation in broiler chickens.

1. The following study was conducted to evaluate the influence of coccidiosis vaccine-induced metabolic stress on the utilisation of minerals in broilers. The starter, grower and finisher phase diets, including macro- and micro minerals at the recommended levels for the breed standards, were fed to chickens between 1 and 39 d of age.2. A total of 486, one-d-old male broilers were randomly distributed into three coccidiosis management programs (CMP) with six replications each. The CMP comprised: monensin sodium (MON), coccidiosis vaccine (VAC), not treated with MON or VAC (CNT).3. No significant differences between CMP were observed for body weight and weight gain among treatments. When compared to the CNT, the VAC program increased feed intake (P < 0.05) between d 1 to 13 and 14 to 26, while FCR worsened in the latter (P < 0.05) and the former (P = 0.05) periods.4. For birds in the MON and VAC programs, tibia bone length at d 13 and bone diameter at d 39 were both enhanced (P < 0.05). Meat yield characteristics were comparable among the CMP.5. Faeces of VAC birds had a lower (P < 0.05) dry matter and ash content than those in CNT program. CMP had no effect on serum or bone mineral concentrations at any point in time. For minerals, Mg, Na, and K faecal excretion was reduced (P < 0.01) as a result of the VAC program at d 13 with a trend at d 26.6. Compared to the CNT, the VAC program decreased the percentage ratio of drip loss (P = 0.08), water holding capacity (P < 0.01) and cooking loss (P < 0.01) in breast meat.7. Overall, the results showed that current broiler industry practices are capable of meeting the mineral needs of broilers vaccinated against coccidiosis.

Broad spectrum post-entry inhibitors of coronavirus replication: Cardiotonic steroids and monensin.

A small molecule screen identified several cardiotonic steroids (digitoxin and ouabain) and the ionophore monensin as potent inhibitors of HCoV-229E, HCoV-OC43, and SARS-CoV-2 replication with EC50s in the low nM range. Subsequent tests confirmed antiviral activity in primary cell models including human nasal epithelial cells and lung organoids. Addition of digitoxin, ouabain, or monensin strongly reduced viral gene expression as measured by both viral protein and RNA accumulation. Furthermore, the compounds acted post virus entry. While the antiviral activity of digitoxin was dependent upon activation of the MEK and JNK signaling pathways but not signaling through GPCRs, the antiviral effect of monensin was reversed upon inhibition of several signaling pathways. Together, the data demonstrates the potent anti-coronavirus properties of two classes of FDA approved drugs that function by altering the properties of the infected cell, rendering it unable to support virus replication.

Effects of a novel direct-fed microbial on growth performance, carcass characteristics, nutrient digestibility, and ruminal morphology of beef feedlot steers.

The effects of a novel direct-fed microbial (DFM) on feedlot performance, carcass characteristics, digestibility, ruminal morphology, and volatile fatty acid (VFA) profile of finishing steers were evaluated. Single-source Angus-crossbred yearling steers (n = 144; initial body weight (BW) = 371 ±â€…19 kg) were used in a randomized complete block design. Steers were blocked by initial BW and randomly assigned to treatments (12 pens/treatment; 4 steers/pen). Treatments included (A) CONTROL (no DFM, tylosin, or monensin, (B) MONTY (monensin sodium [330 mg/animal-daily] and tylosin phosphate [90 mg/animal-daily]), and (C) MONPRO (monensin sodium [same as previous] and Lactobacillus salivarius L28 [1 × 106 CFU/animal-daily]). Treatments were included in a steam-flaked corn-based finisher diet offered once daily using a clean-bunk management for ~149 d. The digestibility assessment was performed from days 70 to 74. Ruminal fluid and rumen tissue samples were collected at the slaughter for VFA profile and papillae morphology analyses, respectively. Data were analyzed using the GLIMMIX procedure of SAS with pen serving as the experimental unit, treatment as fixed effect, and BW block as random effect. Steers offered MONPRO had on average 5.3% less (P < 0.01) dry matter intake (9.56 kg/d) compared with either CONTROL (10.16 kg/d) or MONTY (9.96 kg/d). The carcass-adjusted final BW (613 kg; P = 0.23), overall average daily gain (1.64 kg/d; P = 0.23), and gain-efficiency (0.165; P = 0.61) were not affected by treatments. Steers offered CONTROL had greater (P < 0.01) marbling score and tended (P = 0.06) to have less carcasses grading Select and tended (P = 0.10) to have more carcasses grading Upper-Choice, while other carcass characteristics and liver-abscesses were not affected (P ≥ 0.23) by treatments. The digestibility of nutrients (P ≥ 0.13) and the ruminal VFA profile (P ≥ 0.12) were not affected by treatments. Steers offered MONPRO tended (P = 0.09) to have 16% greater average papillae number compared to other treatments. Yearlings offered finishing diets containing L. salivarius L28 plus monensin did not affect growth performance, digestibility, or ruminal VFA, but reduced feed intake. Carcass quality was negatively affected by treatments, while animals consuming L. salivarius L28 and monensin tended to improve ruminal morphology. Current findings in ruminal morphology and feed intake may warrant further assessment of diets containing L. salivarius L28 on beef cattle food safety aspects.

Antimicrobial resistance is a growing concern to public health and medically important antibiotics have been listed in the Veterinary Feed Directive. Nutritional technologies, such as direct-fed microbials, are being increasingly studied for the development of an effective use on beef cattle production systems. The newly isolated strain of Lactobacillus salivarius L28 has demonstrated pathogenic inhibition of Escherichia coli, Salmonella, and Listeria monocytogenes on in vitro assessments. The potential benefits have warranted the exploration of L. salivarius L28 in a feedlot setting. Single-source Angus-crossbred yearling steers were offered steam-flaked corn-based finishing diets containing no feed additive, or either a combination of tylosin plus monensin or L. salivarius L28 plus monensin. Steers offered L. salivarius L28 plus monensin consumed 5.3% less feed compared with other treatments, while other growth performance variables and the digestibility of nutrients were not affected. Carcasses from cattle supplemented with monensin had slightly lower carcass quality grades than those not supplemented with monensin. Lactobacillus salivarius L28 plus monensin tended to improve steers ruminal morphology. Current findings may warrant further food safety assessments when cattle are offered diets containing L. salivarius L28.

Luminescent reporter cells enable the identification of broad-spectrum antivirals against emerging viruses.

The emerging viruses SARS-CoV-2 and arenaviruses cause severe respiratory and hemorrhagic diseases, respectively. The production of infectious particles of both viruses and virus spread in tissues requires cleavage of surface glycoproteins (GPs) by host proprotein convertases (PCs). SARS-CoV-2 and arenaviruses rely on GP cleavage by PCs furin and subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P), respectively. We report improved luciferase-based reporter cell lines, named luminescent inducible proprotein convertase reporter cells that we employ to monitor PC activity in its authentic subcellular compartment. Using these sensor lines we screened a small compound library in high-throughput manner. We identified 23 FDA-approved small molecules, among them monensin which displayed broad activity against furin and SKI-1/S1P. Monensin inhibited arenaviruses and SARS-CoV-2 in a dose-dependent manner. We observed a strong reduction in infectious particle release upon monensin treatment with little effect on released genome copies. This was reflected by inhibition of SARS-CoV-2 spike processing suggesting the release of immature particles. In a proof of concept experiment using human precision cut lung slices, monensin potently inhibited SARS-CoV-2 infection, evidenced by reduced infectious particle release. We propose that our PC sensor pipeline is a suitable tool to identify broad-spectrum antivirals with therapeutic potential to combat current and future emerging viruses.

Accidental monensin poisoning in goats.

Sodium monensin is the most frequently used ionophore as a growth promoter in ruminant diets. It has numerous benefits; however its toxic effects have also been observed in several animal species. Naturally occurring cases have not yet been reported in goats. This study describes an outbreak of accidental poisoning, characterizing its clinical, laboratory and pathological findings. Thirty-seven of 40 Anglo Nubian goat kids became intoxicated after receiving a diet that was erroneously supplemented with sodium monensin. They ingested an estimated toxic dose between 25 and 39 mg/kg BW. Clinical evolution was monitored (n = 27), followed by serum creatine kinase (CK) and aspartate aminotransferase (AST) activities measurements, and blood gas analysis. Postmortem examinations were performed between 1 and 8 days of evolution (n = 14). Clinical signs began 5 h after ingestion and included reticuloruminal hypomotility, lethargy, anorexia, tachycardia, cardiac arrhythmia, wet cough, pulmonary and tracheal crackles, and serous nasal discharge. The morbidity and lethality rates were 92.5 and 62.1%, respectively. CK and AST activities increased, reaching median values of 10,860 and 1596 U/L, respectively; the hyperchloremic metabolic acidosis was mild. The lesions were characterized by degeneration and necrosis of the cardiac and skeletal muscles, pulmonary congestion and edema, and passive liver congestion. The kids essentially developed cardiomyopathy with left and right congestive heart failures. Unlike in other ruminant species, skeletal muscle functional disability was infrequent. It can be concluded that monensin is toxic to goats and should be used with caution in their diet.

Triepoxide formation by a flavin-dependent monooxygenase in monensin biosynthesis.

Monensin A is a prototypical natural polyether polyketide antibiotic. It acts by binding a metal cation and facilitating its transport across the cell membrane. Biosynthesis of monensin A involves construction of a polyene polyketide backbone, subsequent epoxidation of the alkenes, and, lastly, formation of cyclic ethers via epoxide-opening cyclization. MonCI, a flavin-dependent monooxygenase, is thought to transform all three alkenes in the intermediate polyketide premonensin A into epoxides. Our crystallographic study has revealed that MonCI's exquisite stereocontrol is due to the preorganization of the active site residues which allows only one specific face of the alkene to approach the reactive C(4a)-hydroperoxyflavin moiety. Furthermore, MonCI has an unusually large substrate-binding cavity that can accommodate premonensin A in an extended or folded conformation which allows any of the three alkenes to be placed next to C(4a)-hydroperoxyflavin. MonCI, with its ability to perform multiple epoxidations on the same substrate in a stereospecific manner, demonstrates the extraordinary versatility of the flavin-dependent monooxygenase family of enzymes.

Effects of monensin in supplements for beef cattle in tropical grazing systems during the rainy season.

Two experiments were carried out to evaluate the effect of monensin in supplements for grazing heifers. In experiment I, treatments consisted of protein supplements (low intake - 0.5 kg/animal/day and high intake - 1.0 kg/animal/day) associated or not with monensin. Animal performance, nutrient intake, and digestibility were evaluated. Forty crossbred heifers with an initial body weight (BW) of 213.8 ± 4.5 kg were used. There was no difference in average daily gain between treatments (average of 0.588 kg/animal/day). There was no interaction between monensin and supplements for intake parameters and digestibility. Dry matter (DM) intake was equal between treatments (~ 2% of BW). However, there was a reduction in pasture intake with the high-intake supplement. In experiment II, treatments consisted of two types of supplements (protein or mineral) associated or not with monensin. The variables analyzed were productive performance, ingestive behavior, and thermal comfort, evaluated through the index of thermal stress for cows (ITSC). Forty crossbred heifers with a BW of 159.2 ± 1.3 kg were used. The type of supplement did not influence the performance of the animals. However, monensin promoted greater weight gain in the animals (110 g/animal/day). There was an interaction effect between supplementation and monensin addition on behavioral activities, except for idle time. The inclusion of monensin in the protein supplement reduced the grazing time. The ITSC value influenced the activities of ingestive behavior, and the increase of this index reduced the grazing time in all treatments. Forage quality influences the response to monensin use, and the addition of monensin in supplements for grazing cattle during the rainy season is recommended for forages with high CP content.