Identification of a Novel Dual Inhibitor of Acetylcholinesterase and Butyrylcholinesterase: In Vitro and In Silico Studies.

The enhancement of cholinergic functions via acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibition is considered a valuable therapeutic strategy for the treatment of Alzheimer's disease. This study aimed to evaluate the in vitro effect of ZINC390718, previously filtered using computational approaches, on both cholinesterases and to characterize, using a molecular dynamics (MD) simulation, the possible binding mode of this compound inside the cholinesterase enzymes. The in vitro cytotoxicity effect was also investigated using a primary astrocyte-enriched glial cell culture. ZINC390718 presented in vitro dual inhibitory activity against AChE at a high micromolar range (IC50 = 543.8 µM) and against BuChE (IC50 = 241.1 µM) in a concentration-dependent manner, with greater activity against BuChE. The MD simulation revealed that ZINC390718 performed important hydrophobic and H-bond interactions with the catalytic residue sites on both targets. The residues that promoted the hydrophobic interactions and H-bonding in the AChE target were Leu67, Trp86, Phe123, Tyr124, Ser293, Phe295, and Tyr341, and on the BuChE target, they were Asp70, Tyr332, Tyr128, Ile442, Trp82, and Glu197. The cytotoxic effect of Z390718, evaluated via cell viability, showed that the molecule has low in vitro toxicity. The in vitro and in silico results indicate that ZINC390718 can be used as chemotype for the optimization and identification of new dual cholinesterase inhibitors.

Methanolic Extracts of Chiococca alba in Aedes aegypti Biorational Management: Larvicidal and Repellent Potential, and Selectivity against Non-Target Organisms.

The use of formulations containing botanical products for controlling insects that vector human and animal diseases has increased in recent years. Plant extracts seem to offer fewer risks to the environment and to human health without reducing the application strategy's efficacy when compared to synthetic and conventional insecticides and repellents. Here, we evaluated the potential of extracts obtained from caninana, Chiococca alba (L.) Hitchc. (Rubiaceae), plants as a tool to be integrated into the management of Aedes aegypti, one of the principal vectors for the transmission of arborviruses in humans. We assessed the larvicidal and repellence performance against adult mosquitoes and evaluated the potential undesired effects of the extracts on non-target organisms. We assessed the susceptibility and predatory abilities of the nymphs of Belostoma anurum, a naturally occurring mosquito larva predator, and evaluated the C. alba extract's cytotoxic effects in mammalian cell lines. Our chromatographic analysis revealed 18 compounds, including rutin, naringin, myricetin, morin, and quercetin. The methanolic extracts of C. alba showed larvicidal (LC50 = 82 (72-94) mg/mL) activity without killing or affecting the abilities of B. anurum to prey upon mosquito larvae. Our in silico predictions revealed the molecular interactions between rutin and the AeagOBP1 receptor to be one possible mechanism for the repellent potential recorded for formulations containing C. alba extracts. Low cytotoxicity against mammalian cell lines reinforces the selectivity of C. alba extracts. Collectively, our findings highlight the potential of C. alba and one of its constituents (rutin) as alternative tools to be integrated into the management of A. aegypti mosquitoes.

Potential Action Mechanism and Inhibition Efficacy of Morinda citrifolia Essential Oil and Octanoic Acid against Stagonosporopsis cucurbitacearum Infestations.

The use of plant-based products has been shown to efficiently inhibit fungi-mediated diseases in agricultural crops. Here, we extracted and evaluated the composition of noni, Morinda citrifolia L., essential oil and assessed its activities against Stagonosporopsis cucurbitacearum in Cucumis melo L. Using in silico molecular approaches, potential interactions between the essential oil major components and S. cucurbitacearum tyrosine-tRNA ligase were predicted. Finally, we also measured the potential interference of plant physiology (the stomatal conductance and net photosynthesis) mediated by the application of the M. citrifolia essential oil. Chromatographic analysis revealed that octanoic acid (75.8%), hexanoic acid (12.8%), and isobutyl pent-4-enyl carbonate (3.1%) were the major essential oil compounds. Octanoic acid and noni essential oil, when used as preventive measures, reduce fungal mycelial growth at a concentration of 5 mg/mL without causing significant damage to the treated leaves, which reinforces their efficacies as preventive tools against S. cucurbitacearum. Molecular docking analyses predicted very stable interactions between the major essential oil constituents and S. cucurbitacearum tyrosine-tRNA ligase, suggesting the interference of these plant-based molecules upon enzyme activation. Octanoic acid and M. citrifolia essential oil at concentrations of 20 mg/mL decreased the stomatal conductance and net photosynthesis rate of melon plants, resulting in robust phytotoxicity. Collectively, our findings indicated that despite the phytotoxicity risks at higher concentrations, M. citrifolia essential oil and octanoic acid, have potential as alternative tools for the integrative management of S. cucurbitacearum.

Local administration of p-coumaric acid decreases lipopolysaccharide-induced acute lung injury in mice: In vitro and in silico studies.

Acute lung injury (ALI) remains to cause a high rate of mortality in critically ill patients. It is known that inflammation is a key factor in the pathogenesis of lipopolysaccharide (LPS)-induced ALI, which makes it a relevant approach to the treatment of ALI. In this study, we evaluated the potential of nasally instilled p-coumaric acid to prevent LPS-induced ALI in mice, by evaluating its effects on cellular and molecular targets involved in inflammatory response via in vitro and in silico approaches. Our results demonstrated that p-coumaric acid reduced both neutrophil accumulation and pro-inflammatory cytokine abundance, and simultaneously increased IL-10 production at the site of inflammation, potentially contributing to protection against LPS-induced ALI in mice. In the in vitro experiments, we observed inhibitory effects of p-coumaric acid against IL-6 and IL-8 production in stimulated A549 cells, as well as reactive oxygen species generation by neutrophils. In addition, p-coumaric acid treatment decreased neutrophil adhesion on the TNF-α-stimulated endothelial cells. According to the in silico predictions, p-coumaric acid reached stable interactions with both the ATP-binding site of IKKß as well as the regions within LFA-1, critical for interaction with ICAM-1, thereby suppressing the production of proinflammatory mediators and hindering the neutrophil infiltration, respectively. Collectively, these findings indicate that p-coumaric acid is a promising anti-inflammatory agent that can be used for developing a pharmaceutical drug for the treatment of ALI and other inflammatory disorders.

Essential oil from Negramina (Siparuna guianensis) plants controls aphids without impairing survival and predatory abilities of non-target ladybeetles.

Plant essential oils are regarded as interesting alternative tools to be integrated into the management of pest insects. However, as they generally consist of mixtures of numerous molecules, the physiological basis for their action is unresolved. Here, we evaluated the effects of essential oil of the Neotropical plant Siparuna guianensis Aubl., commonly known as Negramina, against an important pest insect: the green peach aphid Myzus persicae (Sulzer), and also in two non-target natural enemies: the ladybeetle predators Coleomegilla maculata (DeGeer) and Eriopis connexa (Germar). In addition, we conducted a computational docking analysis for predicting the physical interactions between the two Negramina essential oil major constituents: ß-myrcene and 2-undocanone, and the transient receptor potential (TRP) channels as potential binding receptors in the aphid and ladybeetles. As the most important results, Negramina essential oil caused mortality in M. persicae aphids with an LC95 = 1.08 mg/cm2, and also significantly repelled the aphids at concentrations as low as 0.14 mg/cm2. Our computational docking analysis reinforced such selectivity actions as the Negramina essential oil major compounds (i.e., ß-myrcene and 2-undocanone) bound to the TRP channels of M. persicae but not to ladybeetle-related TRP channels. Interestingly, the exposure to the Negramina essential oil did not affect the predatory abilities of C. maculata but increased the abilities of E. connexa to prey upon M. persicae. Collectively, our findings provided a physiological basis for the insecticidal and selectivity potential of Negramina essential oil, reinforcing its potential as a tool to be used in integrated pest control programs.

Spermatogonial stem cell markers and niche in equids.

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and are located in a highly dynamic microenvironment called "niche" that influences all aspects of stem cell function, including homing, self-renewal and differentiation. Several studies have recently identified specific proteins that regulate the fate of SSCs. These studies also aimed at identifying surface markers that would facilitate the isolation of these cells in different vertebrate species. The present study is the first to investigate SSC physiology and niche in stallions and to offer a comparative evaluation of undifferentiated type A spermatogonia (Aund) markers (GFRA1, PLZF and CSF1R) in three different domestic equid species (stallions, donkeys, and mules). Aund were first characterized according to their morphology and expression of the GFRA1 receptor. Our findings strongly suggest that in stallions these cells were preferentially located in the areas facing the interstitium, particularly those nearby blood vessels. This distribution is similar to what has been observed in other vertebrate species. In addition, all three Aund markers were expressed in the equid species evaluated in this study. These markers have been well characterized in other mammalian species, which suggests that the molecular mechanisms that maintain the niche and Aund/SSCs physiology are conserved among mammals. We hope that our findings will help future studies needing isolation and cryopreservation of equids SSCs. In addition, our data will be very useful for studies that aim at preserving the germplasm of valuable animals, and involve germ cell transplantation or xenografts of equids testis fragments/germ cells suspensions.

Comparative modeling of DNA and RNA polymerases from Moniliophthora perniciosa mitochondrial plasmid.

BACKGROUND: The filamentous fungus Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora is a hemibiotrophic Basidiomycota that causes witches' broom disease of cocoa (Theobroma cacao L.). This disease has resulted in a severe decrease in Brazilian cocoa production, which changed the position of Brazil in the market from the second largest cocoa exporter to a cocoa importer. Fungal mitochondrial plasmids are usually invertrons encoding DNA and RNA polymerases. Plasmid insertions into host mitochondrial genomes are probably associated with modifications in host generation time, which can be involved in fungal aging. This association suggests activity of polymerases, and these can be used as new targets for drugs against mitochondrial activity of fungi, more specifically against witches' broom disease. Sequencing and modeling: DNA and RNA polymerases of M. perniciosa mitochondrial plasmid were completely sequenced and their models were carried out by Comparative Homology approach. The sequences of DNA and RNA polymerase showed 25% of identity to 1XHX and 1ARO (pdb code) using BLASTp, which were used as templates. The models were constructed using Swiss PDB-Viewer and refined with a set of Molecular Mechanics (MM) and Molecular Dynamics (MD) in water carried out with AMBER 8.0, both working under the ff99 force fields, respectively. Ramachandran plots were generated by Procheck 3.0 and exhibited models with 97% and 98% for DNA and RNA polymerases, respectively. MD simulations in water showed models with thermodynamic stability after 2000 ps and 300 K of simulation. CONCLUSION: This work contributes to the development of new alternatives for controlling the fungal agent of witches' broom disease.