Ferrostatin-1 mitigates cellular damage in a ferroptosis-like environment in Caenorhabditis elegans.

Although iron (Fe) is the most biologically abundant transition metal, it is highly toxic when it accumulates as Fe2+, forming a labile Fe pool and favoring the Fenton reaction. This oxidative scenario leads to a type of caspase-independent programmed cell death, referred to as ferroptosis, where following processes take place: 1) Fe2+ overload; 2) glutathione peroxidase 4 inactivation; 3) lipid peroxidation and 4) glutathione depletion. The present study sought to evaluate the consequences of Fe2+ administration on ferroptosis induction in Caenorhabditis elegans. We demonstrated higher mortality, increased lipid peroxidation, reduced glutathione peroxidase activity, and morphological damage in dopaminergic neurons upon Fe2+ overload. Pharmacological intervention at the level of lipid peroxidation with ferrostatin-1 (250 µM) mitigated the damage and returned the biochemical parameters to basal levels, revealing the potential of this therapeutical approach. Finally, to assess the relationship between ferroptosis and dopamine in a Parkinsonian background, we evaluated the UA44 worm strain which overexpresses the alpha-synuclein protein in cherry-labeled dopaminergic neurons. We demonstrated that Fe2+ administration reduced lethality associated with similar alterations in biochemical and dopaminergic morphological parameters in wild-type animals. These experiments provide mechanistic-based evidence on the efficacy of a pharmacological approach to mitigate the physiological, biochemical, and morphological consequences of Fe2+ overload. At the same time, they encourage further research on the impact of the combined effects resulting from the genetic background and dopamine signaling in a Parkinsonian phenotype.

Low-dose N-acetyl cysteine prevents paraquat-induced mortality in Caenorhabditis elegans.

Exposure to the herbicide paraquat (PQ; 1,1'-dimethyl-4,4'-bipyridinium dichloride) affects the redox balance of the cell, an effect that can be restored by antioxidants, including N-acetyl cysteine (NAC). One hour of exposure to PQ (0 mM, 10 mM, 50 mM, or 100 mM) dose-dependently increased mortality in Caenorhabditis elegans after exposure (immediate toxicity), while this effect was more evident 24 hours thereafter (delayed toxicity). Importantly, pretreatment with NAC 0.5 mM for one hour partially prevented mortality in the immediate assay, while it had no effect in the delayed test, revealing the importance of long-term studies when evaluating toxicity.

Reduced acute functional tolerance and enhanced preference for ethanol in Caenorhabditis elegans exposed to lead during development: Potential role of alcohol dehydrogenase.

Despite its relative simplicity, the invertebrate Caenorhabditis elegans (C. elegans) has become a powerful tool to evaluate toxicity. Lead (Pb) persistence in the environment and its distinctive characteristic as a neurodevelopmental toxicant determine the potential effects of this metal against challenging events later in life. Additionally, among other psychoactive substances, low to moderate ethanol (EtOH) doses have been pointed out to induce behaviors such as acute functional tolerance (AFT) and drug-induced chemotaxis. In the present study, we aimed to study the impact of early-life Pb exposure on EtOH-induced motivational and stimulant effects in C. elegans by assessing the preference for EtOH and the participation of alcohol dehydrogenase (ADH, sorbitol dehydrogenase -SODH in worms) in the AFT response. Thus, N2 (wild type) and RB2114 (sod-1 -/-) strains developmentally exposed to 24 µM Pb were evaluated in their AFT to 200 mM EtOH alone and in combination with acetaldehyde (ACD). We ascribed the enhanced EtOH-induced AFT observed in the N2 Pb-exposed animals to a reduced ADH functionality as evaluated by both, ADH activity determination and the allyl alcohol test, which altogether suggest excess EtOH accumulation rather than low ACD formation in these animals. Moreover, the Pb-induced preference for EtOH indicates enhanced motivational effects of this drug as a consequence of early-life exposure to Pb, results that resemble our previous reports in rodents and provide a close association between EtOH stimulant and motivational effects in these animals.

Bacterial communities from vagina of dairy healthy heifers and cows with impaired reproductive performance.

Vaginal microenvironment plays a significant role in bovine fertility since its resident microorganisms interact with the host mucosa and constitutes the first barrier against ascending pathogens in the reproductive tract. In this study, the vaginal microbiome of healthy heifers (H) and cows with impaired reproductive performance, metritis complex (MT) or repeat breeders (RB), was assessed using a 16S rRNA gene sequencing approach. Analysis revealed that even though a vaginal microbiological guild (Firmicutes, Bacteroidetes, Proteobacteria, Tenericutes, Fusobacteria and Actinobacteria) was shared among healthy heifers and cows with uterine disease; further analysis at genus level showed significant differences depending on the reproductive health status. The relative abundances of recognized uterine pathogens such as Bacteroidetes, Fusobacterium and Helcococcus were higher in MT when compared with H and RB; therefore, their presence in vagina can be considered as a risk factor for fertility. The present study describes for the first time, the composition of native bacterial communities in the vagina of cows undergoing the repeat breeding syndrome (RBS), and reports an association between this disease and the presence of Porphyromonas and unassigned genera of the Pasteurellaceae family. In addition, this work highlights the bacteria associated with a healthy vagina: genera from the families Lachnospiraceae, Rikenellaceae and the genera Acinetobacter, Bacillus, Oscillospira, CF231 and 5-7NS. Results highlighted herein, signify the potential of the evaluation of the bovine vaginal microbiome to future design therapeutic interventions to improve pregnancy rates however, further research is needed to elucidate the balance of bacterial species resulting in an optimal reproductive health.

Effect of Chitosan Microparticles on the Uterine Microbiome of Dairy Cows with Metritis.

The objective of this study was to evaluate the effect of chitosan microparticles on the uterine microbiome of cows with metritis. Dairy cows with metritis (n = 89) were assigned to 1 of 3 treatments: chitosan microparticles (n = 21), in which the cows received an intrauterine infusion of chitosan microparticles at metritis diagnosis (day 0), day 2, and day 4; ceftiofur (n = 25), in which the cows received a subcutaneous injection of ceftiofur on day 0 and day 3; and no intrauterine or subcutaneous treatment (n = 23). Nonmetritic cows (n = 20) were healthy cows matched with cows with metritis by the number of days postpartum at metritis diagnosis. Uterine swab samples collected on days 0, 3, 6, 9, and 12 were used for 16S rRNA gene sequencing and 16S RNA gene copy number quantification by quantitative PCR. Principal-coordinate analysis showed that the microbiome of the ceftiofur-treated and metritic untreated groups progressed toward that of the nonmetritic group by day 3, whereas that of the chitosan microparticle-treated group remained unchanged. The differences on day 3 were mainly due to a greater relative abundance of Fusobacteria, particularly Fusobacterium, in the chitosan microparticle-treated group than in the ceftiofur-treated and metritic untreated groups. Furthermore, the microbiome of the ceftiofur-treated group became similar to that of the nonmetritic group by day 9, whereas the microbiome of the chitosan microparticle-treated and metritic untreated groups became similar to that of the nonmetritic group only by day 12. The total bacterial 16S rRNA gene counts in the chitosan microparticle-treated group were greater than those in the metritic untreated controls on days 6 and 9, whereas the ceftiofur treatment group was the only group in which the total bacterial 16S rRNA gene count became similar to that in the nonmetritic group by day 12. In summary, chitosan microparticles slowed the progression of the uterine microbiome toward a healthy state, whereas ceftiofur hastened the progression toward a healthy state.IMPORTANCE Third-generation cephalosporins, such as ceftiofur, are commonly used to treat metritis in dairy cows. Chitosan microparticles has been shown to have a broad spectrum of activity in vitro and to be effective against uterine pathogens in vivo; therefore, they have been hailed as a possible alternative to traditional antibiotics. Nonetheless, in the present study, we saw that chitosan microparticle treatment slowed the progression of the uterine microbiome of cows with metritis toward a healthy state, whereas ceftiofur treatment hastened the progression toward a healthy state. Given the lack of an effective alternative to traditional antibiotics and an increased concern about antimicrobial resistance, a greater effort should be devoted to the prevention of metritis in dairy cows.

Characterization of native Escherichia coli populations from bovine vagina of healthy heifers and cows with postpartum uterine disease.

Even though Escherichia coli are common bacteria of the bovine vaginal microbiota, they represent an important pathogen that causes diseases in the reproductive tract and subfertility. However, the actual endometrial virulence profile of E. coli is poorly understood. The present study aims to characterize the phylogenetic structure and virulence potential of native vaginal populations of E. coli from healthy heifers (H), and cows with postpartum uterine diseases (PUD), such as metritis/endometritis (MT) or repeat breeder cows (RB). To this end, the virulence repertoire of 97 E. coli isolates was genotypically and phenotypically assessed. Most of them were assigned to phylogenetic group A (74%), followed by B1 (17%) and D (9%); RB strains were significantly (p < 0.05) more represented by B1. Seven of the 15 evaluated virulence genes (VFG) were detected and the most prevalent were fimH (87%), agn43 (41%) and csgA (35%); while traT (27%), fyuA (11%), hlyA (5%) and kpsMT II (5%) were observed in a lower proportion. Particularly, fyuA was significantly higher (p < 0.05) in MT cows whereas csgA showed the same behavior in PUD animals (p < 0.05). When comparing H and PUD strains, these last ones were associated to positive expression of biofilm, fimbriae curli/cellulose and motility; yet RB strains did not show motility. Vaginal B1 E. coli populations, that possess VFG (fyuA and csgA) as well as the expression of motility, curli fimbriae/cellulose and biofilm, may represent risk factors for endometrial disorders; specifically, those that also, have kpsMT II may have a pathogenic potential for causing the RB syndrome. Future research focusing on the detection of these strains in the vaginal microbiota of cows with postpartum uterine diseases should be done since the control of their presence in vagina could reduce the risk that they access the uterus during the postpartum period.

Antimicrobial resistant Escherichia coli in the reproductive tract microbiota of cows and sows.

Escherichia coli is a natural colonizer of the urogenital mucosa of healthy females; however it is one of the pathogens associated to reproductive failures in cows and sows. A better knowledge about the characteristics of native E. coli will allow us to differentiate them from pathogenic strains. Ninety autochthonous isolates from the reproductive tract of sows and cows were characterized to determine the phylogenetic profile, antibiotic resistance and virulence factors; also, comparisons between different breeding systems were performed. Vaginal colonization of E. coli was statistically higher in cows (57.5%) than sows (23.8%), and most isolates belonged to the phylogenetic group A: 79.69 and 80.77%, respectively; moreover phylo-groups B1 (12.5 and 11.54%) and D (7.81 and 7.69%) were significantly lower; however, none was classified as B2. Positive associations between virulence factors and group D were found. Isolates with antimicrobial susceptibility were associated with group A and the MDR (Multiple Drug Resistance) was related to the porcine source. These results contribute to the knowledge of extra-intestinal E. coli populations; which could affect the reproductive performance of females.

Is there nothing new under the sun? The influence of herbals and pharmacopoeias on ethnobotanical traditions in Albacete (Spain).

ETHNOPHARMACOLOGICAL RELEVANCE: This paper has two overarching aims: (1) presenting the results of studying the Albacete tariff of medicines of 1526 and (2) broadly analyzing the origin and influences of medicinal traditional knowledge in the region of Albacete, Spain. We use historical and modern literature that may have influenced this knowledge. Our primary goal was to determine the ingredients used in the pharmacy in the 16th century CE in Albacete through the analysis of the tariff, and our secondary goal was to investigate until when ingredients and uses present in pharmacy and herbals persisted in later periods. METHODS: The identity of medicines and ingredients was determined by analyzing contemporary pharmacopoeias and classical pharmaceutical references. We analyzed further 21 sources (manuscripts, herbals, and books of medicines, pharmacopoeias, pharmacy inventories, and modern ethnobotanical records) for the presence/absence of ingredients and complex formulations of the tariff. Using factorial and cluster analysis and Bayesian inference applied to evolution models (reversible-jump Markov chain Monte Carlo), we compared textual sources. Finally, we analyzed the medicinal uses of the top 10 species in terms of frequency of citation to assess the dependence of modern ethnobotanical records on Renaissance pharmacy and herbals, and, ultimately, on Dioscorides. RESULTS: In Albacete 1526, we determined 101 medicines (29 simple drugs and 72 compound medicines) comprising 187 ingredients (85% botanical, 7.5% mineral, and 7.5% zoological substances). All composed medicines appear standardized in the pharmacopoeias, notably in the pharmacopoeia of Florence from 1498. However, most were no longer in use by 1750 in the pharmacy, and were completely absent in popular herbal medicine in Albacete 1995 as well as in Alta Valle del Reno (Italy) in 2014. Among the ingredients present in different formulation are the flowers of Rosa gallica, honey (Apis mellifera), the roots of Nardostachys jatamansi, and Convolvulus scammonia, pistils of Crocus sativus, grapes and raisins (Vitis vinifera), rhizomes of Zingiber officinale, bark of Cinnamomum verum, leaves and fruits of Olea europaea, mastic generally of Pistacia lentiscus, and wood of Santalum album. The statistical analysis of sources produces four well-separated clusters (Renaissance Herbals and Pharmacopoeias, Ethnobotany and Folk Medicine, Old phytotherapy, and Modern phytotherapy including Naturopathy) confirming our a priori classification. The clade of Renaissance Herbals and Pharmacopoeias appears separated from the rest in 97% of bootstrapped trees. Bayesian inference produces a tree determined by an initial set of two well-distinct core groups of ingredients: 64, locally used in Mediterranean Europe during centuries; and 45, imported, used in pharmacy during centuries. Complexity reached its maximum in Albacete 1526 and contemporary pharmacopoeias, gradually decreasing over time. The analysis of medicinal uses of the top 10 ingredients showed low coincidence between Dioscorides and different Renaissance herbals or medical treatises and of all of them with ethnobotany in Albacete. CONCLUSIONS: Regarding our question: is there something new under the sun? In some aspects, the answer is "No". The contrast between expensive drugs, highly valued medicines, and unappreciated local wild medicinal plants persists since the Salerno's school of medicine. Old medicine in Mediterranean Europe, as reflected by Albacete 1526 tariff of medicines, involved strict formulations and preferences for certain ingredients despite other ingredients locally available but underappreciated. This confirms the fact that any system of medicine does not get to use all available resources. Ethnobiological records of materia medica, in rural areas of Albacete, describe systems with a high degree of stability and resilience, where the use of local resources, largely wild but also cultivated, is predominant in contrast with the weight of imported exotic products in pharmacy.

Lactic Acid Bacteria Isolated from Bovine Mammary Microbiota: Potential Allies against Bovine Mastitis.

Bovine mastitis is a costly disease in dairy cattle worldwide. As of yet, the control of bovine mastitis is mostly based on prevention by thorough hygienic procedures during milking. Additional strategies include vaccination and utilization of antibiotics. Despite these measures, mastitis is not fully under control, thus prompting the need for alternative strategies. The goal of this study was to isolate autochthonous lactic acid bacteria (LAB) from bovine mammary microbiota that exhibit beneficial properties that could be used for mastitis prevention and/or treatment. Sampling of the teat canal led to the isolation of 165 isolates, among which a selection of ten non-redundant LAB strains belonging to the genera Lactobacillus and Lactococcus were further characterized with regard to several properties: surface properties (hydrophobicity, autoaggregation); inhibition potential of three main mastitis pathogens, Staphylococcus aureus, Escherichia coli and Streptococcus uberis; colonization capacities of bovine mammary epithelial cells (bMEC); and immunomodulation properties. Three strains, Lactobacillus brevis 1595 and 1597 and Lactobacillus plantarum 1610, showed high colonization capacities and a medium surface hydrophobicity. These strains are good candidates to compete with pathogens for mammary gland colonization. Moreover, nine strains exhibited anti-inflammatory properties, as illustrated by the lower IL-8 secretion by E. coli-stimulated bMEC in the presence of these LAB. Full genome sequencing of five candidate strains allowed to check for undesirable genetic elements such as antibiotic resistance genes and to identify potential bacterial determinants involved in the beneficial properties. This large screening of beneficial properties while checking for undesirable genetic markers allowed the selection of promising candidate LAB strains from bovine mammary microbiota for the prevention and/or treatment of bovine mastitis.