Ergot alkaloid consumption alters serotonin receptor-induced vasoactivity in ovine umbilical vasculature.

Consumption of ergot alkaloids during the second half of gestation has been shown to decrease umbilical artery vasoactivity resulting in decreased birth weights. Negative vascular effects of ergot alkaloids are mediated predominantly through serotonergic and adrenergic receptors in other tissues. Vasoactivity of serotonin (5-HT) receptors 5-HT2A and 5-HT1B/1D in umbilical artery and vein from ewes receiving endophyte-infected seed (E + 1.77 mg ergovaline/hd/d) or a control total mixed ration (CON; 0 mg ergovaline/hd/d) tall fescue seed at d-110 and d-133 of gestation was evaluated. Gravid reproduction tracts were collected from ewes. Two-mm sections of umbilical artery and vein were exposed to increasing concentrations of a 5-HT1B/1D agonist and 5-HT2A agonist. The 5-HT1B/1D agonist did not stimulate a contractile response in artery or vein or either gestation time point. 5-HT2A agonist caused large responses in artery with greatest occurring at d-110 and decreasing in magnitude as days of gestation increased (p < 0.05). On d-110 and 133 of gestation, arteries from CON ewes had greater contractile response than arteries collected from E+ ewes (p < 0.05). Veins responded to increasing concentrations of the 5-HT2A agonist. Maximal d-110 vein response was greater than d-133 when exposed to 5-HT2A agonist (p < 0.05). Unlike the artery, veins from E+ ewes had greater d-133 contractile response than CON (p < 0.05). Vascular contractions of umbilical artery and vein are induced by 5-HT2A receptor activity and not 5-HT1B/1D. Umbilical artery 5-HT2A receptor activity was more sensitive to seed treatment and could be responsible for ergot alkaloid-induced intra-uterine growth restriction.

Examining the long-term impacts of ergot alkaloids on fetal heifer development and subsequent growth and reproductive performance.

Consumption of toxic endophyte-infected tall fescue (EI) results in poor reproductive performance in domestic livestock. In this study, the objective was to evaluate the effects of ergovaline exposure during mid-gestation (days 93 through 188 of gestation) on dam performance, the growing female fetus, and the subsequent growth and reproductive performance of the gestationally exposed heifer calves. Pregnant Angus and Simmental-Angus cows were blocked by age (2 to 3, to 7, and >7 y), body weight (BW), and breed; and then randomly assigned to graze either novel endophyte-infected tall fescue (EN; <5% infection rate; n = 27 year 1, n = 16 year 2) or toxic EI (99% infection rate; n = 27 year 1, n = 17 year 2). Weekly BW, body condition scores (BCS), hair coat scores, hair shedding scores (HSS), and blood samples for progesterone (P4) analysis were collected from mid-April through July of 2017 (year 1) and 2018 (year 2). Gestation length, birth weight, placental characteristics, heifer calf growth, onset of puberty, ovarian characteristics, and artificial insemination pregnancy rates were measured. Data were analyzed using the MIXED procedure of SAS. Cows grazing EI pastures had reduced average daily gain, reduced BCS, greater HSS, and decreased P4 concentrations compared to cows on EN pasture (P < 0.01). Birth weights were decreased for heifers whose dams were exposed to EI pastures during their second trimester (P < 0.01). Heifer pregnancy rates were not impacted by EI pasture exposure during gestation for either year of the study. However, a treatment-by-year effect was seen for the pregnancy rate for EI-exposed heifers in year 2; EI-exposed heifers in year 2 had increased pregnancy rates at two of the inseminations. Combined, these data reinforce that consumption of toxic EI during gestation can negatively impact both dam and offspring performance. More studies are needed to evaluate more parameters in an effort to elucidate the possible life-long impacts of ergovaline exposure during gestation.

The U.S. livestock industry incurs over one billion dollars of economic loss every year due to fescue toxicosis, caused by consuming ergot alkaloids produced by an endophytic fungus in some grass species. Identifying means to mitigate the negative effects of fescue toxicosis is needed for U.S. beef producers. Effective treatment for this toxicosis is still needed. The objective of this study was to evaluate the effects of ergovaline exposure during mid-gestation on dam performance, the growing female fetus, and the subsequent growth and reproductive performance of the gestationally exposed heifer calves. We identified specific phenotype traits that undergo developmental programming in utero in response to fescue toxicosis. However, measurements of growth and reproductive performance were not altered by ergot exposure.

Impact of endophyte-infected tall fescue seed consumption on endocrine changes associated with intake regulation and post-absorptive metabolism in growing steers.

Fescue toxicosis is a syndrome occurring from the consumption of endophyte-infected tall fescue and results in substantial economic losses to the beef industry primarily from reduced growth accompanied by decreased dry matter intake (DMI); however, the associations characterizing this reduction in DMI have yet to be elucidated. The objective of this experiment was to identify endocrine changes associated with intake regulation post-consumption of endophyte-infected tall fescue seed (E+). Twelve Holstein steers were stratified by body weight and assigned to 1 of 3 treatments (n=4): 0 ppm ergovaline (ERV), 1.8 ppm ERV, or 2.7 ppm ERV. Treatments were achieved by combining differing proportions of ground E+ and non-endophyte-infected tall fescue seed. Steers were adapted to their diets for 7 d followed by a 7 d DMI collection period. Within treatment, steers were assigned to a sampling day (d 16 or d 17). Blood samples were collected every 20 min for 8 h, beginning 1 h before feeding. Intake data was analyzed using the MIXED procedure of SAS 9.4 (SAS Inst. Inc., Cary, NC) with treatment, day, and the interaction as fixed effects. Hormone and metabolite data were analyzed with the fixed effect of treatment, time, and the interaction including time as a repeated measure and orthogonal contrasts. Dry matter intake was linearly decreased with increasing ERV in the diet (P < 0.001). Insulin and leptin concentrations exhibited a quadratic effect (P = 0.018 and P = 0.005) with insulin concentrations highest for the 2.7 ppm treatment and leptin concentrations highest for the 1.8 ppm treatment. No differences were detected for active ghrelin or ß-hydroxybuytrate concentrations among treatment groups. Further, steers consuming both the 1.8 and 2.7 ppm ERV treatments had lower prolactin concentrations compared to the 0 ppm treatment (quadratic, P= 0.019). Glucose concentrations had a tendency for a linear increase as ERV concentrations increased (P = 0.091). A treatment × time interaction (P = 0.002) was noted in NEFA concentrations, with the 1.8 ppm ERV treatment showing increased pre-feeding concentrations, and the 2.7 ppm ERV treatment exhibiting elevated NEFA concentrations as time post-feeding progressed. The results suggest that E+ consumption reduces intake likely through alterations in intake-related hormones and post-absorptive metabolism and contributes to our current understanding of E+ effects on intake reduction while providing avenues for future research.

Hepatic transcript profiling in beef cattle: Effects of feeding endophyte-infected tall fescue seeds.

The objective of our study was to evaluate the effect of endophyte-infected tall fescue (E+) seeds intake on liver tissue transcriptome in growing Angus × Simmental steers and heifers through RNA-seq analysis. Normal weaned calves (~8 months old) received either endophyte-free tall fescue (E-; n = 3) or infected tall fescue (E+; n = 6) seeds for a 30-d period. The diet offered was ad libitum bermudagrass (Cynodon dactylon) hay combined with a nutritional supplement of 1.61 kg (DM basis) of E+ or E- tall fescue seeds, and 1.61 kg (DM basis) of energy/protein supplement pellets for a 30-d period. Dietary E+ tall fescue seeds were included in a rate of 20 µg of ergovaline/kg BW/day. Liver tissue was individually obtained through biopsy at d 30. After preparation and processing of the liver samples for RNA sequencing, we detected that several metabolic pathways were activated (i.e., upregulated) by the consumption of E+ tall fescue. Among them, oxidative phosphorylation, ribosome biogenesis, protein processing in endoplasmic reticulum and apoptosis, suggesting an active mechanism to cope against impairment in normal liver function. Interestingly, hepatic protein synthesis might increase due to E+ consumption. In addition, there was upregulation of "thermogenesis" KEGG pathway, showing a possible increase in energy expenditure in liver tissue due to consumption of E+ diet. Therefore, results from our study expand the current knowledge related to liver metabolism of growing beef cattle under tall fescue toxicosis.

Anti-herbivory defences delivered by Epichloë fungal endophytes: a quantitative review of alkaloid concentration variation among hosts and plant parts.

BACKGROUND AND AIMS: In the subfamily Poöideae (Poaceae), certain grass species possess anti-herbivore alkaloids synthesized by fungal endophytes that belong to the genus Epichloë (Clavicipitaceae). The protective role of these symbiotic endophytes can vary, depending on alkaloid concentrations within specific plant-endophyte associations and plant parts. METHODS: We conducted a literature review to identify articles containing alkaloid concentration data for various plant parts in six important pasture species, Lolium arundinaceum, Lolium perenne, Lolium pratense, Lolium multiflorum|Lolium rigidum and Festuca rubra, associated with their common endophytes. We considered the alkaloids lolines (1-aminopyrrolizidines), peramine (pyrrolopyrazines), ergovaline (ergot alkaloids) and lolitrem B (indole-diterpenes). While all these alkaloids have shown bioactivity against insect herbivores, ergovaline and lolitrem B are harmful for mammals. KEY RESULTS: Loline alkaloid levels were higher in the perennial grasses L. pratense and L. arundinaceum compared to the annual species L. multiflorum and L. rigidum, and higher in reproductive tissues than in vegetative structures. This is probably due to the greater biomass accumulation in perennial species that can result in higher endophyte mycelial biomass. Peramine concentrations were higher in L. perenne than in L. arundinaceum and not affected by plant part. This can be attributed to the high within-plant mobility of peramine. Ergovaline and lolitrem B, both hydrophobic compounds, were associated with plant parts where fungal mycelium is usually present, and their concentrations were higher in plant reproductive tissues. Only loline alkaloid data were sufficient for below-ground tissue analyses and concentrations were lower than in above-ground parts. CONCLUSIONS: Our study provides a comprehensive synthesis of fungal alkaloid variation across host grasses and plant parts, essential for understanding the endophyte-conferred defence extent. The patterns can be understood by considering endophyte growth within the plant and alkaloid mobility. Our study identifies research gaps, including the limited documentation of alkaloid presence in roots and the need to investigate the influence of different environmental conditions.

Ruminal ergovaline and volatile fatty acid dynamics: Association with poor performance and a key growth regulator in steers grazing toxic tall fescue.

Fescue toxicosis (FT) is produced by an ergot alkaloid (i.e., ergovaline [EV])-producing fungus residing in toxic fescue plants. Associations between EV, decreased weight gain and ruminal volatile fatty acids are unclear. Feces, rumen fluid, and blood were collected from 12 steers that grazed non-toxic (NT) or toxic (E +) fescue for 28 days. The E + group exhibited decreased propionate (P), increased acetate (A), and increased ruminal A:P ratio, with similar trends in feces. Plasma GASP-1 (G-Protein-Coupled-Receptor-Associated-Sorting-Protein), a myostatin inhibitor, decreased (day 14) only in E + steers. Ergovaline was present only in E + ruminal fluid and peaked on day 14. The lower ruminal propionate and higher A:P ratio might contribute to FT while reduced GASP-1 might be a new mechanism linked to E + -related weight gain reduction. Day 14 ergovaline zenith likely reflects ruminal adaptations favoring EV breakdown and its presence only in rumen points to local, rather than systemic effects.

A Rapid and Sensitive Stability-Indicating Eco-Friendly HPTLC Assay for Fluorescence Detection of Ergotamine.

Eco-friendly liquid chromatographic methods for measuring ergotamine (EGT) are scant in the published database. Accordingly, the goal of the current study was to develop a high-performance thin-layer chromatography (HPTLC) method for fluorescence detection of EGT in commercially available tablets. This approach was based on the application of ethyl alcohol-water (80:20 v/v) as the eco-friendly eluent mixture. The fluorescence detection of EGT was carried out at 322 nm. The greenness score of the present approach was evaluated by "Analytical GREENness (AGREE)" technology. The present approach for measuring EGT in the 25-1000 ng band-1 range was linear. The present assay for fluorescence detection of EGT was validated successfully by ICH guidelines for various parameters. The method was found to be rapid, sensitive, eco-friendly, and stability-indicating. The computed AGREE index for the current strategy was 0.84, displaying outstanding greenness features. The present methodology successfully separated the EGT degradation products under forced-degradation circumstances, exhibiting its stability-indicating qualities and selectivity. An amount of 99.33% of EGT was found in commercial formulations, indicating the validity of the current method for pharmaceutical analysis of EGT in commercial products. The results showed that EGT in commercial products might be regularly measured by the existing method.

Duration of ergovaline exposure influences serotonin-mediated vasoactivity of bovine mesenteric vasculature.

Ergovaline (ERV), produced in toxic endophyte-infected tall fescue, causes potent vasoconstriction of bovine peripheral and visceral vasculature. Ergovaline acts as both an agonist and an antagonist in bovine gut blood vessels through serotonin (5-HT) receptors and it appears that the type of action could be influenced by the extent of ERV exposure. Because it was unclear how the duration of ERV exposure influences 5-HT-mediated vasoactivity, experiments were designed to evaluate how simultaneous or prior ERV exposure influenced 5-HT-mediated vasoactivity of mesenteric artery (MA) and vein (MV) segments from Holstein steers (N = 10). Vessels were incubated in Krebs-Henseleit buffer containing 0, 0.01, or 0.1 µM ERV for 24 h prior to the 5-HT dose-response or exposed to fixed concentrations of 0, 0.01, or 0.1 µM ERV simultaneously during the 5-HT dose-response. Vessels were suspended in chambers of a multimyograph containing Krebs-Henseleit buffer and equilibrated to 1 g tension for 90 min. Vessels were exposed to increasing concentrations of 5-HT (5 × 10-8 M to 1 × 10-4 M) every 15 min and contractile responses were normalized as a percentage of the maximum contractile response induced by 120 mM KCl reference addition. Two-way analysis of variance was used to separately analyze data for each vessel type and duration of exposure using the MIXED procedure of SAS. When 5-HT concentration increased from 5 × 10-8 to 1 × 10-6 M, simultaneous addition of 0.1 µM ERV increased (P < 0.01) the contractile response of MV compared with additions of 0 and 0.01 µM ERV. At 1 × 10-4 M 5-HT, the simultaneous presence of 0.01 and 0.1 µM ERV decreased (P < 0.01) the contractile response of both MA and MV compared with 0 µM ERV addition. As 5-HT concentrations increased, the contractile response increased (P < 0.01) in both MA and MV with no previous ERV exposure, but decreased in MA and MV with 24 h prior exposure to 0.01 and 0.1 µM ERV. These data demonstrate that the duration of ERV exposure influences 5-HT-mediated vasoconstriction and likely vasorelaxation in bovine mesenteric vasculature. If ERV and 5-HT exposure occur simultaneously, ERV can act as a partial agonist of 5-HT-mediated vasoconstriction. If 5-HT exposure occurs after blood vessels have had prior ERV exposure, it appears that 5-HT may induce vasorelaxation of blood vessels. More research is needed to identify cellular and molecular mechanisms involved with 5-HT-mediated vasoactivity.

Consumption of ergot alkaloids found in endophyte-infected tall fescue can lead to symptoms of fescue toxicosis, such as vasoconstriction, in ruminant livestock species. Ergovaline is one of the primary ergot alkaloids responsible for causing vasoconstriction when toxic varieties of fescue are consumed. It has been previously shown that ergovaline causes vasoconstriction by interacting with vascular serotonin receptors in cattle and sheep. Depending on when ergovaline exposure occurs, ergovaline can function as an agonist (stimulant) or antagonist (inhibitor) of vascular activity. However, it is unclear how the duration of ergovaline exposure affects vasoconstriction caused by serotonin. Experiments were conducted using the bovine mesenteric artery and mesenteric vein that were exposed to either 0, 0.01, or 0.1 µM ergovaline for 24-h prior to serotonin additions or simultaneously with serotonin additions. Maximum contractile response data were recorded using a multimyograph system and normalized as a percentage of the contractile response produced by the reference compound, KCl. The results of these experiments demonstrated that the duration of ergovaline exposure influences serotonin-mediated vasoconstriction and possibly vasorelaxation in bovine mesenteric vasculature. If ergovaline and serotonin exposure occur simultaneously, ergovaline can act as an agonist or antagonist of serotonin-mediated vasoconstriction. If serotonin exposure occurs after prior ergovaline exposure, serotonin can induce vasorelaxation of blood vessels. Understanding how complex interactions between ergovaline and serotonin occur and affect vascular function will aid in the development of strategies to mitigate sustained vasoconstriction caused during fescue toxicosis.

Use of 3D applicator for intranasal microneedle arrays for combinational therapy in migraine.

The objective of current research work was to fabricate dissolving microneedles combining ergotamine and caffeine for synergistic action using controlled release kinetics with better permeability. The method of preparation for microneedles utilized multiple emulsion (w/o/w) approach by solvent-diffusion-evaporation process wherein the nano-emulsion of ergotamine and caffeine prepared using PLGA polymer and PVA as a stabilizer. The PLGA nanospheres were further loaded in polymer matrix of PVA and PVP K-90 and the final mixture poured in sterile silicon molds of microneedles. The PLGA nanospheres exhibited particle size in narrow range of 280.34 ± 6.61 to 416.0 ± 9.67 nm and good colloidal stability with negative zeta potential ranging between -19.08 ± 8.77 to -22.49 ± 8.09 mV. Higher entrapment efficiency (86.21 ± 4.52 %) for ergotamine and controlled release pattern (49.79 ± 4.16 % at 48 h) displayed by PLGA nanospheres. Similarly, the dissolving microneedles loaded with PLGA nanospheres showed controlled release pattern for in-vitro and ex-vivo drug release studies with 52.01 ± 5.71 % for ERM and 87.04 ± 2.44 % for CFE at 48 h whereas ex-vivo release studies illustrated similar results of 51.08 ± 3.56 % for ERM and 69.2 ± 2.16 % for CFE. The anti-hyperalgesic capability of microneedles was verified by the acetic acid writhing test, and the non-toxicity of synthetic microneedles was confirmed by histopathology and serotonin toxicity studies. The novel 3D applicator effectively delivered the microneedle array into the nasal cavity for systemic action. Therefore, the fabricated rapid dissolving microneedles combining two drugs ergotamine and caffeine with use of 3D applicator proved to be a coherent technique for intranasal delivery of ergotamine in the treatment of migraine.

Finding alternatives to 5-fluorouracil: application of ensemble-based virtual screening for drug repositioning against human thymidylate synthase.

5-fluorouracil and analogs are used in the treatment of many solid tumours. However, there are many cases of resistance and high toxicity associated with 5-fluorouracil chemotherapy. Repurposing FDA drugs against human thymidylate synthase revealed a number of FDA drugs that have a potential to be further developed for the treatment of various cancers for which 5-fluorouracil and analogs have been used for chemotherapy. Four FDA drugs prioritized for further validation included Erismodegib, Irinotecan, Conivaptan and Ergotamine. The role of water in mediating drug interactions and its contribution to the total binding energy was also shown. MM-PBSA calculations revealed that the binding affinity was the lowest for the hTS-Ergotamine complex (-66.702 ± 1.807 kJ/mol) suggesting moderate inhibition despite a large energetic contribution from van der Waal interactions (-190.889 ± 1.027 kJ/mol).Communicated by Ramaswamy H. Sarma.

Self-Assembled Lipid Polymer Hybrid Nanoparticles Using Combinational Drugs for Migraine Via Intranasal Route.

The objective of the current research study was to formulate the PEGylated lipid polymer hybrid nanoparticles of ergotamine and caffeine for intranasal administration with higher entrapment efficiency, better permeability, desirable controlled release pattern, and significant brain uptake in animal studies. A single-step nanoprecipitation method was employed in the processing of self-assembled hybrid nanoparticles constituting polymer PLGA, lipids soya lecithin, and DPPC with PEGylation using polyethylene glycol (PEG-2000). The optimal lipid/polymer weight ratio of 15% w/w showed lower particle size of 239.46 ± 2.31 nm with good colloidal stability depicted by zeta potential (- 18.36 ± 6.59 mV), higher entrapment efficiency of 86.88 ± 1.67%, and controlled release profile when evaluated for in vitro and ex vivo studies as 97.12 ± 2.79% and 75.13 ± 5.62% release, respectively, for 48 h. The formulation showed long-term serum stability when incubated in bovine serum albumin and displayed high brain uptake (4.35-fold) offering significant permeability in the brain post-intranasal administration via olfactory route. Histopathological investigations and serotonin toxicity studies in animals confirmed the safe and non-toxic nature of the formulation while the acetic acid writhing test proved the anti-hyperalgesic activity. The PEGylated lipid polymer hybrid nanoparticles of ergotamine and caffeine showed synergistic activity with efficacious higher anti-migraine potential.

Effects of Heating, Pelleting, and Feed Matrix on Apparent Concentrations of Cereal Ergot Alkaloids in Relation to Growth Performance and Welfare Parameters of Backgrounding Beef Steers.

As the contamination of cereal grains with ergot has been increasing in Western Canada, studies were undertaken to evaluate the impacts of heating (60, 80, 120, or 190 °C) alone or in combination with pelleting on concentrations of ergot alkaloids. Fifteen samples of ergot-contaminated grain from Alberta and Saskatchewan were assayed for R and S epimers of six alkaloids (ergocryptine, ergocristine, ergocornine, ergometrine, ergosine, and ergotamine) using HPLC MS/MS. Five samples with distinct alkaloid profiles were then selected for heating and pelleting studies. Heating resulted in a linear increase (p < 0.05) of total R and total S epimers with increasing temperature, although some individual R epimers were stable (ergometrine, ergosine, ergotamine). Pelleting also increased (p < 0.05) concentrations of total R and total S epimers detected, although ergometrine concentration decreased (p < 0.05) after pelleting. A feeding study arranged in a 2 × 2 factorial structure used 48 backgrounding Angus-cross steers fed four different diets: (1) Control Mash (CM, no added ergot), (2) Control Pellet (CP), (3) Ergot Mash (EM), or (4) Ergot Pellet (EP). Pelleting heated the ergot to 90−100 °C under 4 bars pressure, but the ergot used in the feeding study was not otherwise heated. Alkaloid concentrations of EM and EP varied by up to 1.1 mg/kg depending on the feed matrix assayed. No differences among treatments were noted for growth performance, feed intake, feed conversion, concentrations of serum prolactin and haptoglobin, hair cortisol, or in temperatures of extremities measured by infrared thermography. The only negative impacts of ergot alkaloids were on blood parameters indicative of reduced immune function or chronic inflammation. Pelleting did not heighten the negative clinical outcomes of ergot, although alkaloid concentrations of pelleted feed increased depending on the matrix assayed. It was hypothesized that the heat and pressure associated with pelleting may enhance the recovery of alkaloids from pelleted feed.

Ion mobility-mass spectrometry to extend analytical performance in the determination of ergot alkaloids in cereal samples.

This work evaluates the potential of ion mobility spectrometry (IMS) to improve the analytical performance of current liquid chromatography-mass spectrometry (LC-MS) workflows applied to the determination of ergot alkaloids (EAs) in cereal samples. Collision cross section (CCS) values for EA epimers are reported for the first time to contribute to their unambiguous identification. Additionally, CCS values have been inter-laboratory cross-validated and compared with CCS values predicted by machine-learning models. Slight differences were observed in terms of CCS values for ergotamine, ergosine and ergocristine and their corresponding epimers (from 3.3 to 4%), being sufficient to achieve a satisfactory peak-to-peak resolution for their unequivocal identification. A LC-travelling wave ion mobility (TWIM)-MS method has been developed for the analysis of EAs in barley and wheat samples. Signal-to-noise ratio (S/N) was improved between 2.5 and 4-fold compared to the analog LC-TOF-MS method. The quality of the extracted ion chromatograms was also improved by using IMS.

In Silico Screening of Novel TMPRSS2 Inhibitors for Treatment of COVID-19.

COVID-19, a pandemic caused by the virus SARS-CoV-2, has spread globally, necessitating the search for antiviral compounds. Transmembrane protease serine 2 (TMPRSS2) is a cell surface protease that plays an essential role in SARS-CoV-2 infection. Therefore, researchers are searching for TMPRSS2 inhibitors that can be used for the treatment of COVID-19. As such, in this study, based on the crystal structure, we targeted the active site of TMPRSS2 for virtual screening of compounds in the FDA database. Then, we screened lumacaftor and ergotamine, which showed strong binding ability, using 100 ns molecular dynamics simulations to study the stability of the protein-ligand binding process, the flexibility of amino acid residues, and the formation of hydrogen bonds. Subsequently, we calculated the binding free energy of the protein-ligand complex by the MM-PBSA method. The results show that lumacaftor and ergotamine interact with residues around the TMPRSS2 active site, and reached equilibrium in the 100 ns molecular dynamics simulations. We think that lumacaftor and ergotamine, which we screened through in silico studies, can effectively inhibit the activity of TMPRSS2. Our findings provide a basis for subsequent in vitro experiments, having important implications for the development of effective anti-COVID-19 drugs.

Phyto-Compounds from a Rather Poisonous Plant, Strychnos nuxvomica, Show High Potency Against SARS-CoV-2 RNA-Dependent RNA Polymerase.

BACKGROUND: The establishment of strategy to inhibit the virus replication is an attractive means in combating SARS-CoV-2 infection. OBJECTIVE: We studied phyto-compounds from Strychnos nux-vomica (a poisonous plant) against SARS-CoV-2 RNA-dependent RNA polymerase by computational methods. METHODS: Molecular docking, molecular dynamics (MD) simulation and energetics calculations were employed to elucidate the role of the phyto-compounds. RESULTS: Ergotamine with a binding free energy of -14.39 kcal/mol showed a promising capability in terms of binding affinity and the interaction to conserved motifs, especially the SDD signature sequence. The calculated dissociation constants for ATP, ergotamine, isosungucine and sungucine were 12 µM, 0.072 nM, 0.011 nM and 0.152 nM, respectively. The exhibited kd by these phyto-compounds reflected tens of thousands fold potency as compared to ATP. The binding free energies of sungucine and isosungucine were much lower (-13.93 and -15.55 kcal/mol, respectively) compared to that of ATP (-6.98 kcal/mol). CONCLUSION: Sharing the same binding location as that of ATP and having high binding affinities, Ergotamine, Isosungucine, Sungucine and Strychnine N-oxide could be effective in controlling the SARS-CoV-2 virus replication by blocking the ATP and inhibiting the enzyme function.

Using On-Farm Monitoring of Ergovaline and Tall Fescue Composition for Horse Pasture Management.

Central Kentucky horse pastures contain significant populations of tall fescue (Schedonorus arundinacea (Schreb.) Dumort) infected with an endophyte (Epichloë coenophialum (Morgan-Jones and Gams) Bacon and Schardl) known to produce several ergot alkaloids, with ergovaline in the highest concentration. While most classes of horses are not adversely affected by average levels of ergovaline in pastures, late term pregnant mares have a low tolerance to ergovaline and the related ergot alkaloids. Endophyte-infected tall fescue has been known to cause prolonged gestation, thickened placenta, dystocia, agalactia, and foal and mare mortality. The University of Kentucky Horse Pasture Evaluation Program utilizes ergovaline and endophyte testing, as well as pasture species composition, to calculate ergovaline in the total diet in broodmare pastures. This data is used to develop detailed management recommendations for individual pastures. Application of these recommendations has led to reduced tall fescue toxicity symptoms on these farms, as well as improved pasture management and improved forage quality and quantity.

The Usage of Ergot (Claviceps purpurea (fr.) Tul.) in Obstetrics and Gynecology: A Historical Perspective.

In the past centuries consumption of bread made of ergot-infected flour resulted in mass poisonings and miscarriages. The reason was the sclerotia of Claviceps purpurea (Fr.) Tul.-a source of noxious ergot alkaloids (ergotamine and ergovaline). The authors have searched the 19th century medical literature in order to find information on the following topics: dosage forms of drugs based on ergot and their application in official gynecology and obstetrics. The authors also briefly address the relevant data from the previous periods as well as the 20th century research on ergot. The research resulted in a conclusion that applications of ergot in gynecology and obstetrics in the 19th century were limited to controlling excessive uterine bleeding and irregular spasms, treatment of fibrous tumors of the uterus, and prevention of miscarriage, abortion, and amenorrhoea. The most common dosage forms mentioned in the works included in our review were the following: tinctures, water extracts (Wernich's and Squibb's watery extract of ergot), pills, and powders. The information documented in this paper will be helpful for further research and helpful in broadening the understanding of the historical application of the described controversial crude drugs. Ergot alkaloids were widely used in obstetrics, but in modern times they are not used in developed countries anymore. They may, however, play a significant role in developing countries where, in some cases, they can be used as an anti-hemorrhage agent during labor.

Determination of the Main Ergot Alkaloids and Their Epimers in Oat-Based Functional Foods by Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry.

An ultra-high performance liquid chromatography coupled to tandem mass spectrometry method is proposed for the determination of the major ergot alkaloids (ergometrine, ergosine, ergotamine, ergocornine, ergokryptine, ergocristine) and their epimers (ergometrinine, ergosinine, ergotaminine, ergocorninine, ergokryptinine, and ergocristinine) in oat-based foods and food supplements. A modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure was applied as sample treatment, reducing the consumption of organic solvent and increasing sensitivity. This method involved an extraction with acetonitrile and ammonium carbonate (85:15, v/v) and a clean-up step based on dispersive solid-phase extraction, employing a mixture of C18/Z-Sep+ as sorbents. Procedural calibration curves were established and limits of quantification were below 3.2 µg/kg for the studied compounds. Repeatability and intermediate precision (expressed as RSD%) were lower than 6.3% and 15%, respectively, with recoveries ranging between 89.7% and 109%. The method was applied to oat-based products (bran, flakes, flour, grass, hydroalcoholic extracts, juices, and tablets), finding a positive sample of oat bran contaminated with ergometrine, ergosine, ergometrinine, and ergosinine (total content of 10.7 µg/kg).

Occurrence of Ergot Alkaloids in Barley and Wheat from Algeria.

The natural occurrence of six major ergot alkaloids, ergometrine, ergosine, ergotamine, ergocornine, ergokryptine and ergocristine, as well as their corresponding epimers, were investigated in 60 cereal samples (barley and wheat) from Algeria. Ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) and a QuEChERS extraction method were used for sample analysis. The results revealed that 12 out of 60 samples (20%) were contaminated with ergot alkaloids. Wheat was the most contaminated matrix, with an incidence of 26.7% (8 out of 30 samples). The concentration of total ergot alkaloids ranged from 17.8 to 53.9 µg/kg for barley and from 3.66 to 76.0 µg/kg for wheat samples. Ergosine, ergokryptine and ergocristine showed the highest incidences in wheat, while ergometrine was the most common ergot in barley.

A critical review of analytical methods for ergot alkaloids in cereals and feed and in particular suitability of method performance for regulatory monitoring and epimer-specific quantification.

Cereals and feed contaminated with ergot alkaloids (EAs) have been of concern for several decades. Nowadays, analysis of EAs is focused on ergometrine, ergotamine, ergosine, ergocristine, ergocryptine (a mixture of α- and ß-isomers) and ergocornine and their related -inine epimers as listed in the European Commission Recommendation 2012/154/EU. Liquid chromatography with fluorescence detection has been used for quantification of EAs for decades whilst LC-MS has become the work-horse for quantification of EAs in the last decade. However, in LC-MS analysis matrix effects of different magnitudes exist for each EA epimer, especially ergometrine/ergometrinine, even after different clean-up procedures. This leads to an underestimation or overestimation of EAs levels. Moreover, isotopic labelled standards for EAs are still not available in the market. This review aims to provide background information on different analytical methods, discuss their advantages and disadvantages and possible advancement. Moreover, the method performance requirements to support forthcoming regulations are also discussed.