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During a research program to identify new cholinesterase inhibitors of natural origin, two new 7,8-didehydroprotoberberine alkaloids ( 1 and 2 ) and nine known compounds ( 3 - 11 ) were isolated from the capsules of the common ornamental poppy, Papaver setiferum (previously P. pseudo-orientale ). Despite their reported instability, the 7,8-didehydroprotoberberines isolated herein appeared relatively stable, particularly as their trifluoroacetic acid salts. The spatial distributions of the isolated alkaloids were also analyzed using desorption electrospray ionization imaging mass spectrometry. The alkaloids were localized predominantly within the walls and vascular bundles of the capsules, with the highest relative abundances occurring in the lower half of the capsules toward the peduncle. The relative abundances of the alkaloids were also compared across plant development stages. Although most alkaloids did not show clear patterns in their concentration across development stages, the concentration of suspected oxidation products clearly spiked upon plant death. Finally, all isolated natural products were screened for inhibitory activities against a panel of cholinesterases, from both human and animal sources. These studies identified several competitive inhibitors of cholinesterases with potency in the low micromolar range ( 1 - 4 , 6 , 7 ), offering new lead compounds for the development of cholinesterase inhibitory drugs.

Cholinesterases (ChEs) show increased activities in patients with Alzheimer's disease, and remain one of the main therapeutic targets for treatment of this neurodegenerative disorder. A library of organoruthenium(II) complexes was prepared to investigate the influence of their structural elements on inhibition of ChEs, and on another pharmacologically important group of enzymes, glutathione S-transferases (GSTs). Two groups of organoruthenium(II) compounds were considered: (i) organoruthenium(II) complexes with p -cymene as an arene ligand, and (ii) organoruthenium(II) carbonyl complexes as CO-releasing molecules. Eight organoruthenium complexes were screened for inhibitory activities against ChEs and GSTs of human and animal origins. Some compounds inhibited all of these enzymes at low micromolar concentrations, while others selectively inhibited either ChEs or GSTs. This study demonstrates the importance of the different structural elements of organoruthenium complexes for their inhibitory activities against ChEs and GSTs, and also proposes some interesting compounds for further preclinical testing as ChE or GST inhibitory drugs.

Aegerolysins are small lipid-binding proteins particularly abundant in fungi. Aegerolysins from oyster mushrooms interact with an insect-specific membrane lipid and, together with MACPF proteins produced by the same organism, form pesticidal pore-forming complexes. The specific interaction with the same membrane lipid was recently demonstrated for nigerolysin A2 (NigA2), an aegerolysin from Aspergillus niger . In Aspergillus species, the aegerolysins were frequently found as secreted proteins, indicating their function in fungal defense. Using immunocytochemistry and live-cell imaging we investigated the subcellular localization of the nigerolysins A in A. niger , while their secretion was addressed by secretion prediction and Western blotting. We show that both nigerolysins A are leaderless proteins that reach the cell exterior by an unconventional protein secretion. NigA proteins are evenly distributed in the cytoplasm of fungal hyphae. A detailed bioinformatics analysis of Aspergillus aegerolysins suggests that the same function occurs only in a limited number of aegerolysins. From alignment, analysis of chromosomal loci, orthology, synteny, and phylogeny it follows that the same or a similar function described for pairs of pesticidal proteins of Pleurotus sp. can be expected in species of the subgenus Circumdati , section Nigri , series Nigri , and some other species with adjacent pairs of putative pesticidal proteins.

Many Arctic biomes, which are populated with abundant and diverse microbial life, are under threat: climate change and warming temperatures have raised concerns about diversity loss and possible emergence of pathogenic microorganisms. At present, there is little information on the occurrence of Arctic virulence-associated phenotypes. In this study we worked with 118 strains of bacteria (from 10 sampling sites in the Arctic region, located in Greenland and the Svalbard Archipelago) isolated using R2A medium. These strains belong to 4 phyla and represent 36 different bacterial genera. Phenotypic resistance to 8 clinically important antimicrobials (ampicillin, chloramphenicol, ciprofloxacin, cefotaxime, erythromycin, imipenem, kanamycin, and tetracycline) and thermotolerance range were determined. In addition, a screening of all isolates on blood agar media and erythrocytes suspension of bovine and sheep erythrocytes for virulence-linked hemolytic activity was performed. Although antimicrobial resistance profiles varied among the isolates, they were consistent within bacterial families and genera. Interestingly, a high number of isolates (83/104) were resistant to the tested concentration of imipenem (4 mg/L). In addition, one third of the isolates showed hemolytic activity on blood agar, however, in only 5% of the isolates hemolytic activity was also observed in the cell extracts when added to erythrocyte suspensions for 60 min. The observed microbial phenotypes contribute to our understanding of the presence of virulence-associated factors in the Arctic environments, while highlighting the potential risks associated with changes in the polar areas in the light of climate change., (Copyright © 2020 Mogrovejo, Perini, Gostinčar, Sepčić, Turk, Ambrožič-Avguštin, Brill and Gunde-Cimerman.)

Covering: Published between 1974 up to 2018Inhibition of cholinesterases is a common approach for the management of several disease states. Most notably, cholinesterase inhibitors are used to alleviate the symptoms of neurological disorders like dementia and Alzheimer's disease and treat myasthenia gravis and glaucoma. Historically, most drugs of natural origin have been isolated from terrestrial sources and inhibitors of cholinesterases are no exception. However, the last 50 years have seen a rise in the quantity of marine natural products with close to 25 000 reported in the scientific literature. A number of marine natural products with potent cholinesterase inhibitory properties have also been reported; isolated from a variety of marine sources from algae to ascidians. Representing a diverse range of structural classes, these compounds provide inspirational leads that could aid the development of therapeutics. The current paper aims to, for the first time, comprehensively summarize the literature pertaining to cholinesterase inhibitors derived from marine sources, including the first papers published in 1974 up to 2018. The review does not report bioactive extracts, only isolated compounds, and a specific focus lies on compounds with reported dose-response data. In vivo and mechanistic data is included for compounds where this is reported. In total 185 marine cholinesterase inhibitors and selected analogs have been identified and reported and some of the compounds display inhibitory activities comparable or superior to cholinesterase inhibitors in clinical use.

Six Monascus purpureus (red mould) strains were cultivated on brown rice and millet as substrates. They underwent strain selection for high lovastatin and pigment production, and low citrinin mycotoxin production, with particular reference to potential for millet as substrate. For most of these strains, substrate dry matter loss was 54-60% on rice and 46-48% on millet, although the 'MOPU GS1' strain showed 18% and 17% dry matter loss, respectively. 'MOPU GS1' was also the only strain with detectable levels of lovastatin (1.3 and 1.6 mg lovastatin/g substrate dry weight [dw], respectively) and citrinin under the limit of detection. In the other strains, citrinin varied from 0.3 to 18.2 mg/g substrate dw. Among the six strains, 'EBY3' provided high pigment production when grown on rice, although it produced 1.1 mg citrinin/g substrate dw. Millet showed good potential as an alternative substrate to rice, due to higher lovastatin and lower citrinin production; however, rice was the better substrate for production of M. purpureus pigments.

Cholinesterases (ChE), the enzymes whose primary function is the hydrolysis of choline esters, are widely expressed throughout the nature. Although they have already been found in plants and microorganisms, including ascomycete fungi, this study is the first report of ChE-like activity in fungi of the phylum Basidiomycota. This activity was detected in almost a quarter of the 45 tested aqueous fungal extracts. The ability of these extracts to hydrolyse acetylthiocholine was about ten times stronger than the hydrolytic activity towards butyrylthiocholine and propionylthiocholine. In-gel detection of ChE-like activity with acetylthiocholine indicated a great variability in the characteristics of these enzymes which are not characterized as vertebrate-like based on (i) differences in inhibition by excess substrate, (ii) susceptibility to different vertebrate acetylcholinesterase and butyrylcholinesterase inhibitors, and (iii) a lack of orthologs using phylogenetic analysis. Limited inhibition by single inhibitors and multiple activity bands using in-gel detection indicate the presence of several ChE-like enzymes in these aqueous extracts. We also observed inhibitory activity of the same aqueous mushroom extracts against insect acetylcholinesterase in 10 of the 45 samples tested; activity was independent of the presence of ChE-like activity in extracts. Both ChE-like activities with different substrates and the ability of extracts to inhibit insect acetylcholinesterase were not restricted to any fungal family but were rather present across all included Basidiomycota families. This study can serve as a platform for further research regarding ChE activity in mushrooms., Competing Interests: The authors have declared that no competing interests exist.

The extensive production of zinc oxide (ZnO) nanomaterials (NMs) may result in high environmental zinc burdens. Honeybees need to have special concern due to their crucial role in pollination. Our previous study indicated that low concentrations of ZnO NMs, corresponding to 0.8 mg Zn/mL, have a neurotoxic potential for honeybees after a 10-day oral exposure. Present study was designed to investigate the effect of a short, dietary exposure of honeybees to ZnO NMs at concentrations 0.8-8 mg Zn/mL on consumption rate, food preference, and two enzymatic biomarkers-a stress-related glutathione S-transferase (GST) and the neurotoxicity biomarker acetylcholinesterase (AChE). Consumption rate showed a tendency toward a decrease feeding with the increasing concentrations of ZnO NMs. None of Zn NMs concentrations caused alterations in mortality rate and in the activities of brain GST and AChE. To investigate if there is an avoidance response against Zn presence in food, 24-h two-choice tests were performed with control sucrose diet versus sucrose suspensions with different concentrations of ZnO NMs added. We demonstrated that honeybees prefer ZnO NMs ZnO NMs containing suspensions, even at highest Zn concentrations tested, compared with the control diet. This indicates that they might be able to perceive the presence of ZnO NMs in sucrose solution. Because honeybees feed frequently the preference towards ZnO NMs might have a high impact on their survival when exposed to these NMs.

Background: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus., Results: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli., Conclusions: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.

The aim was to investigate the toxicity of selected metal oxide nanoparticles (MO-NPs) on the brine shrimp Artemia salina, by evaluating mortality and behavioural and biochemical responses. Larvae were exposed to tin(IV) oxide (stannic oxide (SnO2)), cerium(IV) oxide (CeO2) and iron(II, III) oxide (Fe3O4) NPs for 48 h in seawater, with MO-NP suspensions from 0.01 to 1.0 mg/mL. Mortality and behavioural responses (swimming speed alteration) and enzymatic activities of cholinesterase, glutathione-S-transferase and catalase were evaluated. Although the MO-NPs did not induce any mortality of the larvae, they caused changes in behavioural and biochemical responses. Swimming speed significantly decreased in larvae exposed to CeO2 NPs. Cholinesterase and glutathione-S-transferase activities were significantly inhibited in larvae exposed to SnO2 NPs, whereas cholinesterase activity significantly increased after CeO2 NP and Fe3O4 NP exposure. Catalase activity significantly increased in larvae exposed to Fe3O4 NPs. In conclusion, swimming alteration and cholinesterase activity represent valid endpoints for MO-NP exposure, while glutathione-S-transferase and catalase activities appear to be NP-specific.

We report on the screening for biological activities of organic extracts from seven strains that represent four varieties of the fungus Aureobasidium pullulans, that is A. pullulans var. melanogenum, A. pullulans var. pullulans, A. pullulans var. subglaciale and A. pullulans var. namibiae. We monitored haemolysis, cytotoxicity, antioxidant capacity and growth inhibition against three bacterial species. The haemolytic activity of A. pullulans var. pullulans EXF-150 strain was due to five different haemolytically active fractions. Extracts from all of the other varieties contained at least one haemolytically active fraction. Short-term exposure of cell lines to these haemolytically active organic extracts resulted in more than 95% cytotoxicity. Strong antioxidant capacity, corresponding to 163.88 μg ascorbic acid equivalent per gram of total solid, was measured in the organic extract of the strain EXF-3382, obtained from A. pullulans var. melanogenum, isolated from the deep sea. Organic extracts from selected varieties of A. pullulans exhibited weak antibacterial activities.

Proteins with membrane-attack complex/perforin (MACPF) domains are found in almost all kingdoms of life, and they have a variety of biological roles, including defence and attack, organism development, and cell adhesion and signalling. The distribution of these proteins in fungi appears to be restricted to some Pezizomycotina and Basidiomycota species only, in correlation with another group of proteins with unknown biological function, known as aegerolysins. These two protein groups coincide in only a few species, and they might operate in concert as cytolytic bi-component pore-forming agents. Representative proteins here include pleurotolysin B, which has a MACPF domain, and the aegerolysin-like protein pleurotolysin A, and the very similar ostreolysin A, which have been purified from oyster mushroom (Pleurotus ostreatus). These have been shown to act in concert to perforate natural and artificial lipid membranes with high cholesterol and sphingomyelin content. The aegerolysin-like proteins provide the membrane cholesterol/sphingomyelin selectivity and recruit oligomerised pleurotolysin B molecules, to create a membrane-inserted pore complex. The resulting protein structure has been imaged with electron microscopy, and it has a 13-meric rosette-like structure, with a central lumen that is ~4-5 nm in diameter. The opened transmembrane pore is non-selectively permeable for ions and smaller neutral solutes, and is a cause of cytolysis of a colloid-osmotic type. The biological significance of these proteins for the fungal life-style is discussed.

APS12-2 is one in a series of synthetic analogs of the polymeric alkylpyridinium salts isolated from the marine sponge Reniera sarai. As it is a potential candidate for treating non small cell lung cancer (NSCLC), we have studied its possible toxic and lethal effects in vivo. The median lethal dose (LD(50)) of APS12-2 in mice was determined to be 11.5mg/kg. Electrocardiograms, arterial blood pressure and respiratory activity were recorded under general anesthesia in untreated, pharmacologically vagotomized and artificially ventilated rats injected with APS12-2. In one group, the in vivo effects of APS12-2 were studied on nerve-evoked muscle contraction. Administration of APS12-2 at a dose of 8mg/kg caused a progressive reduction of arterial blood pressure to a mid-circulatory value, accompanied by bradycardia, myocardial ischemia, ventricular extrasystoles, and second degree atrio-ventricular block. Similar electrocardiogram and arterial blood pressure changes caused by APS12-2 (8mg/kg) were observed in animals pretreated with atropine and in artificially ventilated animals, indicating that hypoxia and cholinergic effects do not play a crucial role in the toxicity of APS12-2. Application of APS12-2 at sublethal doses (4 and 5.5mg/kg) caused a decrease of arterial blood pressure, followed by an increase slightly above control values. We found that APS12-2 causes lysis of rat erythrocytes in vitro, therefore it is reasonable to expect the same effect in vivo. Indeed, hyperkalemia was observed in the blood of experimental animals. Hyperkalemia probably plays an important role in APS12-2 cardiotoxicity since no evident changes in histopathology of the heart were found. However, acute lesions were observed in the pulmonary vessels of rats after application of 8mg/kg APS12-2. Predominant effects were dilation of interalveolar blood vessels and lysis of aggregated erythrocytes within their lumina., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Unlabelled: Two new large poly-1,3-dodecylpyridinium salts, APS12 and APS12-2 of 12.5- and 14.7-kDa size, respectively, were synthesised and tested for their pore-forming and transfection capabilities in HEK 293 and undifferentiated mouse ES cells using patch-clamp recording, Ca(2+) imaging and flow cytometry. Polymerisation reactions were enhanced by microwaves, and the product sizes were controlled by altering the irradiation time. This method can also be applied to obtain polymers with variable linking chains as shown by the preparation of poly-(1,3-octylpyridinium) salt of 11.9-kDa size. Molecular weights of the final products were determined using ESIMS analysis, which also indicated the products to be amongst the largest macro-cycles ever recorded, up to a 900-membered ring. Anti-bacterial, haemolytic and anti-acetylcholinesterase activities were also reported for the two dodecyl pyridinium polymers. These biological activities are characteristic to the structurally related marine toxin, poly-APS., Electronic Supplementary Material: The online version of this article (doi:10.1007/s12154-010-0036-4) contains supplementary material, which is available to authorized users.