Screening of Brazilian basidiomycetes for antimicrobial activity

A total of 103 isolates of basidiomycetes, representing 84 species from different Brazilian ecosystems, were evaluated for their antifungal and antibacterial activity in a panel of pathogenic and non-pathogenic microorganisms. Tissue plugs of the fruiting bodies were cultivated in liquid media and the whole culture extracted with ethyl acetate. Crude extracts from Agaricus cf. nigrecentulus, Agrocybe perfecta, Climacodon pulcherrimus, Gloeoporus thelephoroides, Hexagonia hydnoides, Irpex lacteus, Leucoagaricus cf. cinereus, Marasmius cf. bellus, Marasmius sp., Nothopanus hygrophanus, Oudemansiella canarii, Pycnoporus sanguineus, Phellinus sp., and Tyromyces duracinus presented significant activity against one or more of the target microorganisms. Eight isolates were active only against bacteria while three inhibited exclusively the growth of fungi. Two extracts presented wide antimicrobial spectrum and were active against both fungi and bacteria. Differences in the bioactivity of extracts obtained from isolates from the same species were observed.

Antibacterial activity of extracts and neolignans from Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck

The evaluation of the activity of the aqueous and ethyl acetate extracts of the leaves of Piper regnellii was tested against gram-positive and gram-negative bacteria. The aqueous extractdisplayed a weak activity against Staphylococcus aureus and Bacillus subtilis with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 1000 µg/ml. The ethyl acetate extract presented a good activity against S. aureus and B. subtilis with MIC and MBC at 15.62 µg/ml. In contrast to the relative low MICs for gram-positive bacteria, gram-negative bacteria were not inhibited by the extracts at concentrations < 1000 mg/ml. The ethyl acetate extract was fractionated on silica gel into nine fractions. The hexane and chloroform fractions were active against S. aureus (MIC at 3.9 µg/ml) and B. subtilis (MIC at 3.9 and 7.8 µg/ml, respectively). Using bioactivity-directed fractionation, the hexane fraction was rechromatographed to yield the antimicrobial compounds 1, 2, 5, and 6identified as eupomatenoid-6, eupomatenoid-5, eupomatenoid-3, and conocarpan, respectively. The pure compounds 1 and 2 showed a good activity against S. aureus with MIC of 1.56 µg/ml and 3.12 µg/ml, respectively. Both compounds presented MIC of 3.12 µg/ml against B. subtilis. The pure compound 6 named as conocarpan was quite active against S. aureus and B. subtilis with MIC of 6.25 µg/ml. The antibacterial properties of P. regnellii justify its use in traditional medicine for the treatment of wounds, contaminated through bacteria infections.

Biophysical characterization of the interaction of endotoxins with hemoglobins.

Bacterial endotoxin (lipopolysaccharide, LPS) is the major component of the outer leaflet of the outer membrane in gram-negative bacteria. During severe infections, bacteria may reach the blood circuit of humans, and endotoxins may be released from the bacteria due to cell division or cell death. In particular enterobacterial forms of LPS represent extremely strong activator molecules of the human immune system causing a rapid induction of cytokine production in monocytes and macrophages. Various mammalian blood proteins have been documented to display LPS binding activities mediating normally decreasing effects in the biological activity of LPS. In more recent studies, the essential systemic oxygen transportation protein hemoglobin (Hb) has been shown to amplify LPS-induced cytokine production on immune cells. The mechanism responsible for this effect is poorly understood. Here, we characterize the interaction of hemoglobin with LPS by using biophysical methods. The data presented, revealing the changes of the type and size of supramolecular aggregates of LPS in the presence of Hb, allow a better understanding of the hemoglobin-induced increase in bioactivity of LPS.

Comparison of visual preservation after transplantation of BDNF or GDNF secreting mesenchymal stem cells in glaucomatous rat eyes

Purpose:To functionally and morphologically characterize the retina and optic nerve after transplantation of Brain-derived neurotrophic factor (BDNF) and Glial-derived neurotrophic factor (GDNF) secreting mesenchymal stem cells (MSCs) into glaucomatous rat eyes. Methods:Chronic ocular hypertension (COH) was induced in Brown Norway rats. Lentiviral constructs were used to transduce rat MSCs to produce BDNF, GDNF, or green fluorescent protein (GFP). The fellow eyes served as internal controls. Two days following COH induction, eyes received intravitreal injections of transduced MSCs. Electroretinography was performed to assess retinal function. Tonometry was performed throughout the experiment to monitor IOP. 42 days after MSC transplantation, rats were euthanized and the eyes and optic nerves were prepared for analysis. Results:Increased expression and secretion of BDNF and GDNF from lentiviral-transduced MSCs was verified using ELISA, and a bioactivity assay. Ratio metric analysis (COH eye/ Internal control eye response) of the Max combined response A-Wave showed animals with BDNF-MSCs (23.35 ± 5.15%, p=0.021) and GDNF-MSCs (28.73 ± 3.61%, p=0.025) preserved significantly more visual function than GFP-MSC treated eyes MSCs (18.05 ± 5.51%). Animals receiving BDNF-MSCs also had significantly better B-wave (33.80 ± 7.19%) and flicker ERG responses (28.52 ± 10.43%) than GFP-MSC treated animals (14.06 ± 12.67%; 3.52 ± 0.07%, respectively). Animals receiving GDNF-MSC transplants tended to have better function than animals with GFP-MSC transplants, but were not statistically significant (p=0.057 and p=0.0639). Conclusions:Mesenchymal stem cells are an excellent source of cells for autologous transplantation for the treatment of neurodegenerative diseases. We have demonstrated that lentiviral- transduced MSCs can survive following transplantation and preserve visual function in glaucomatous eyes. These results suggest that MSCs may be an ideal cellular vehicle for delivery of specific neurotrophic factors to the retina.

Cocoa polyphenols and inflammatory markers of cardiovascular disease.

Epidemiological studies have demonstrated the beneficial effect of plant-derived food intake in reducing the risk of cardiovascular disease (CVD). The potential bioactivity of cocoa and its polyphenolic components in modulating cardiovascular health is now being studied worldwide and continues to grow at a rapid pace. In fact, the high polyphenol content of cocoa is of particular interest from the nutritional and pharmacological viewpoints. Cocoa polyphenols are shown to possess a range of cardiovascular-protective properties, and can play a meaningful role through modulating different inflammatory markers involved in atherosclerosis. Accumulated evidence on related anti-inflammatory effects of cocoa polyphenols is summarized in the present review.

SwissTargetPrediction: a web server for target prediction of bioactive small molecules.

Bioactive small molecules, such as drugs or metabolites, bind to proteins or other macro-molecular targets to modulate their activity, which in turn results in the observed phenotypic effects. For this reason, mapping the targets of bioactive small molecules is a key step toward unraveling the molecular mechanisms underlying their bioactivity and predicting potential side effects or cross-reactivity. Recently, large datasets of protein-small molecule interactions have become available, providing a unique source of information for the development of knowledge-based approaches to computationally identify new targets for uncharacterized molecules or secondary targets for known molecules. Here, we introduce SwissTargetPrediction, a web server to accurately predict the targets of bioactive molecules based on a combination of 2D and 3D similarity measures with known ligands. Predictions can be carried out in five different organisms, and mapping predictions by homology within and between different species is enabled for close paralogs and orthologs. SwissTargetPrediction is accessible free of charge and without login requirement at http://www.swisstargetprediction.ch.

SwissBioisostere: a database of molecular replacements for ligand design.

The SwissBioisostere database (http://www.swissbioisostere.ch) contains information on molecular replacements and their performance in biochemical assays. It is meant to provide researchers in drug discovery projects with ideas for bioisosteric modifications of their current lead molecule, as well as to give interested scientists access to the details on particular molecular replacements. As of August 2012, the database contains 21 293 355 datapoints corresponding to 5 586 462 unique replacements that have been measured in 35 039 assays against 1948 molecular targets representing 30 target classes. The accessible data were created through detection of matched molecular pairs and mining bioactivity data in the ChEMBL database. The SwissBioisostere database is hosted by the Swiss Institute of Bioinformatics and available via a web-based interface.

Endurance training enhances vasodilation induced by nitric oxide in human skin.

Résumé Il a été démontré que l'exercice physique modifiait le contrôle de la thermorégulation cutané, ce qui se manifeste par une augmentation de la perfusion de la microcirculation de la peau. Pour une même augmentation de température, ce phénomène est plus important chez les sportifs d'endurance que chez les sujets sédentaires. Dans cette étude, nous posons l'hypothèse qu'une composante de cette adaptation peut provenir d'une plus haute capacité des vaisseaux sanguins à répondre à un stimulus vasodilatateur. Pour la tester, nous avons recruté des hommes sains, non fumeurs, soit entraînés (surtout sport d'endurance) ou sédentaires que nous avons partagé en deux classes d'âges (18-35 ans [jeunes] et >50 ans[âgés]). Le flux sanguin cutané était mesuré par un laser-Doppler au niveau de la peau de l'avant-bras. Nous avons alors mesuré la vasodilatation obtenue par les stimuli suivant : Iontophorèse à l'acétylcholine (ACh, un vasodilatateur dépendant de l'endothélium), iontophorèse au nitroprussiate de sodium (SNP, un donneur d'oxyde nitrique) et par libération d'une interruption momentanée du flux artériel huméral (hyperémie réactive). Chez les sujets entraînés, l'effet de l'hyperémie réactive et de l'ACh n'ont pas montré de différence. Par contre, l'augmentation de la perfusion, suivant la iontophorèse de SNP, exprimé en unité de perfusion (PU), était plus importante chez les sujets entraînés que chez les sujets sédentaires (jeunes: 398±54 vs 350±87, p<0.05; âgés: 339±72 vs 307±66, p<0.05). Pour conclure, l'entraînement d'endurance augmente l'effet vasodilatateur de l'oxyde nitrique de la microcirculation cutanée humaine, au moins au niveau de la peau de l'avant-bras. Ces observations ont un intérêt physiologique considérable au vu des résultats d'études récentes qui montrent que le NO sert d'intermédiaire dans la vasodilatation cutanée produite par un stress thermique. Donc, l'augmentation de la bioactivité du NO dans la microcirculation cutanée pourrait être un des mécanismes par lequel l'entraînement physique modifierait le contrôle de la thermorégulation du flux sanguin cutané. Abstract Endurance training modifies the thermoregulatory control of skin blood flow, as manifested by a greater augmentation of skin perfusion for the same increase in core temperature in athletes, in comparison with se-dentary subjects. In this study, we tested the hypothesis that a component of this adaptation might reside in a higher ability of cutaneous blood vessels to respond to vasodilatory stimuli. We recruited healthy nonsmoking males, either endurance trained or sedentary, in two different age ranges (18-35 y and >50 y). Skin blood flow was measured in the forearm skin, using a laser Doppler imager, allowing to record the vasodilatory responses to the following stimuli: iontophoresis of acetylcholine (an endothelium-dependent vasodilator), iontophoresis of sodium nitroprusside (a nitric oxide donor), and release of a temporary interruption of arterial inflow (reactive hyperemia). There was no effect of training on reactive hyperemia or the response to acetylcholine. In contrast, the increase in perfusion following the iontophoresis of sodium nitroprusside, ex-pressed in perfusion units, was larger in trained than in sedentary subjects (younger: 398±54 vs 350±87, p<0.05; older 339±72 vs 307±66, p<0.05). In conclusion, endurance training enhances the vasodilatory effects of nitric oxide in the human dermal microcirculation, at least in forearm skin. These observations have considerable physiologic interest in view of recent data indicating that nitric oxide mediates in part the cutaneous vasodilation induced by heat stress in humans. Therefore, the augmentation of nitric oxide bioactivity in the dermal microcirculation might be one mechanism whereby endurance training modifies the thermoregulatory control of skin blood flow.

Positive effects of an angiotensin II type 1 receptor antagonist in Camurati-Engelmann disease: a single case observation.

Camurati-Engelmann disease is characterized by hyperostosis of the long bones and the skull, muscle atrophy, severe limb pain, and progressive joint contractures in some patients. It is caused by heterozygous mutations in the transforming growth factor β1 (TGFβ1) believed to result in improper folding of the latency-associated peptide domain of TGFβ1 and thus in increased or deregulated bioactivity. Losartan, an angiotensin II type 1 receptor antagonist, has been found to downregulate the expression of TGFβ type 1 and 2 receptors. Clinical trials with losartan have shown a benefit in Marfan syndrome, while trials are underway for Duchenne muscular dystrophy and other myopathies associated with TGFβ1 signaling. We hypothesized that due to its anti-TGFβ1 activity, losartan might be beneficial in Camurati-Engelmann disease. This report concerns a boy who presented at age 13 years with severe limb pain and difficulty in walking. Clinical and radiographic evaluation results were compatible with Camurati-Engelmann disease and the diagnosis was confirmed by mutation analysis (c.652C > T [p.Arg218Cys]). The boy underwent an experimental treatment with losartan at a dosage of 50 mg/day, orally. During the treatment period of 18 months, the intensity and frequency of limb pain decreased significantly (as shown by a pain diary), and muscle strength improved, allowing the boy to resume walking and climbing stairs. No obvious side effects were observed. We cautiously conclude that TGFβ1 inhibition with losartan deserves further evaluation in the clinical management of Camurati-Engelmann disease.

Root growth of tomato seedlings intensified by humic substances from peat bogs

Peats are an important reserve of humified carbon in terrestrial ecosystems. The interest in the use of humic substances as plant growth promoters is continuously increasing. The objective of this study was to evaluate the bioactivity of alkaline soluble humic substances (HS), humic (HA) and fulvic acids (FA) isolated from peats with different decomposition stages of organic matter (sapric, fibric and hemic) in the Serra do Espinhaço Meridional, state of Minas Gerais. Dose-response curves were established for the number of lateral roots growing from the main plant axis of tomato seedlings. The bioactivity of HA was greatest (highest response in lateral roots at lowest concentration) while FA did not intensify root growth. Both HS and HA stimulated root hair formation. At low concentrations, HS and HA induced root hair formation near the root cap, a typical hormonal imbalance effect in plants. Transgenic tomato with reporter gene DR5::GUS allowed the observation that the auxin-related signalling pathway was involved in root growth promotion by HA.

VEGF is a mediator of retinal pigment epithelium permeability leading to photoreceptor cell death in light-induced retinal degeneration : gene therapy strategy to prevent AMD-disorders

ABSTRACTIn normal tissues, a balance between pro- and anti-angiogenic factors tightly controls angiogenesis. Alterations of this balance may have pathological consequences. For instance, concerning the retina, the vascular endothelial growth factor (VEGF) is a potent pro-angiogenic factor, and has been identified has a key player during ocular neovascularization implicated in a variety of retinal diseases. In the exudative form (wet-form) of age-related macular degeneration (AMD), neovascularizations occurring from the choroidal vessels are responsible for a quick and dramatic loss of visual acuity. In diabetic retinopathy and retinopathy of prematurity, sprouting from the retinal vessels leads to vision loss. Furthermore, the aging of the population, the increased- prevalence of diabetes and the better survival rate of premature infants will lead to an increasing rate of these conditions. In this way, anti-VEGF strategy represents an important therapeutic target to treat ocular neovascular disorders.In addition, the administration of Pigmented Epithelial growth factor, a neurotrophic and an anti- angiogenic factor, prevents photoreceptor cell death in a model of retinal degeneration induced by light. Previous results analyzing end point morphology reveal that the light damage (LD) model is used to mimic retinal degenerations arising from environmental insult, as well as aging and genetic disease such as advanced atrophic AMD. Moreover, light has been identified as a co-factor in a number of retinal diseases, speeding up the degeneration process. This protecting effect of PEDF in the LD retina raises the possibility of involvement of the balance between pro- and anti-angiogenic factors not only for angiogenesis, but also in cell survival and maintenance.The aim of the work presented here was to evaluate the importance of this balance in neurodegenerative processes. To this aim, a model of light-induced retinal degeneration was used and characterized, mainly focusing on factors simultaneously controlling neuron survival and angiogenesis, such as PEDF and VEGF.In most species, prolonged intense light exposure can lead to photoreceptor cell damage that can progress to cell death and vision loss. A protocol previously described to induce retinal degeneration in Balb/c mice was used. Retinas were characterized at different time points after light injury through several methods at the functional and molecular levels. Data obtained confirmed that toxic level of light induce PR cell death. Variations were observed in VEGF pathway players in both the neural retina and the eye-cup containing the retinal pigment epithelium (RPE), suggesting a flux of VEGF from the RPE towards the neuroretina. Concomitantly, the integrity of the outer blood-retinal-barrier (BRB) was altered, leading to extravascular albumin leakage from the choroid throughout the photoreceptor layer.To evaluate the importance of VEGF during light-induced retinal degeneration process, a lentiviral vector encoding the cDNA of a single chain antibody directed against all VEGF-A isoforms was developed (LV-V65). The bioactivity of this vector to block VEGF was validated in a mouse model of laser-induced choroidal neovascularization mediated by VEGF upregulation. The vector was then used in the LD model. The administration of the LV-V65 contributed to the maintenance of functional photoreceptors, which was assessed by ERG recording, visual acuity measurement and histological analyses. At the RPE level, the BRB integrity was preserved as shown by the absence of albumin leakage and the maintenance of RPE cell cohesion.These results taken together indicate that the VEGF is a mediator of light induced PR degeneration process and confirm the crucial role of the balance between pro- and anti-angiogenic factors in the PR cell survival. This work also highlights the prime importance of BRB integrity and functional coupling between RPE and PR cells to maintain the PR survival. VEGF dysregulation was already shown to be involved in wet AMD forms and our study suggests that VEGF dysregulation may also occur at early stages of AMD and could thus be a potential therapeutic target for several RPE related diseases.RESUMEDans les différents tissues de l'organisme, l'angiogenèse est strictement contrôlée par une balance entre les facteurs pro- et anti-angiogéniques. Des modifications survenant dans cette balance peuvent engendrer des conséquences pathologiques. Par exemple, concernant la rétine, le facteur de croissance de l'endothélium vasculaire (VEGF) est un facteur pro-angiogénique important. Ce facteur a été identifié comme un acteur majeur dans les néovascularisations oculaires et les processus pathologiques angiogéniques survenant dans l'oeil et responsables d'une grande variété de maladies rétiniennes. Dans la forme humide de la dégénérescence maculaire liée à l'âge (DMLA), la néovascularisation choroïdienne est responsable de la perte rapide et brutale de l'acuité visuelle chez les patients affectés. Dans la rétinopathie diabétique et celle lié à la prématurité, l'émergence de néovaisseaux rétiniens est la cause de la perte de la vision. Les néovascularisations oculaires représentent la principale cause de cécité dans les pays développés. De plus, l'âge croissant de la population, la progression de la prévalence du diabète et la meilleure survie des enfants prématurés mèneront sans doute à l'augmentation de ces pathologies dans les années futures. Dans ces conditions, les thérapies anti- angiogéniques visant à inhiber le VEGF représentent une importante cible thérapeutique pour le traitement de ces pathologies.Plusieurs facteurs anti-angiogéniques ont été identifiés. Parmi eux, le facteur de l'épithélium pigmentaire (PEDF) est à la fois un facteur neuro-trophique et anti-angiogénique, et l'administration de ce facteur au niveau de la rétine dans un modèle de dégénérescence rétinienne induite par la lumière protège les photorécepteurs de la mort cellulaire. Des études antérieures basées sur l'analyse morphologique ont révélé que les modifications survenant lors de la dégénération induite suite à l'exposition à des doses toxiques de lumière représente un remarquable modèle pour l'étude des dégénérations rétiniennes suite à des lésions environnementales, à l'âge ou encore aux maladies génétiques telle que la forme atrophique avancée de la DMLA. De plus, la lumière a été identifiée comme un co-facteur impliqué dans un grand nombre de maladies rétiniennes, accélérant le processus de dégénération. L'effet protecteur du PEDF dans les rétines lésées suite à l'exposition de des doses toxiques de lumière suscite la possibilité que la balance entre les facteurs pro- et anti-angiogéniques soit impliquée non seulement dans les processus angiogéniques, mais également dans le maintient et la survie des cellules.Le but de ce projet consiste donc à évaluer l'implication de cette balance lors des processus neurodégénératifs. Pour cela, un modèle de dégénération induite par la lumière à été utilisé et caractérisé, avec un intérêt particulier pour les facteurs comme le PEDF et le VEGF contrôlant simultanément la survie des neurones et l'angiogenèse.Dans la plupart des espèces, l'exposition prolongée à une lumière intense peut provoquer des dommages au niveau des cellules photoréceptrices de l'oeil, qui peut mener à leur mort, et par conséquent à la perte de la vision. Un protocole préalablement décrit a été utilisé pour induire la dégénération rétinienne dans les souris albinos Balb/c. Les rétines ont été analysées à différents moments après la lésion par différentes techniques, aussi bien au niveau moléculaire que fonctionnel. Les résultats obtenus ont confirmé que des doses toxiques de lumière induisent la mort des photorécepteurs, mais altèrent également la voie de signalisation du VEGF, aussi bien dans la neuro-rétine que dans le reste de l'oeil, contenant l'épithélium pigmentaire (EP), et suggérant un flux de VEGF provenant de ΙΈΡ en direction de la neuro-rétine. Simultanément, il se produit une altération de l'intégrité de la barrière hémato-rétinienne externe, menant à la fuite de protéine telle que l'albumine, provenant de la choroïde et retrouvée dans les compartiments extravasculaires de la rétine, telle que dans la couche des photorécepteurs.Pour déterminer l'importance et le rôle du VEGF, un vecteur lentiviral codant pour un anticorps neutralisant dirigée contre tous les isoformes du VEGF a été développé (LV-V65). La bio-activité de ce vecteur a été testé et validée dans un modèle de laser, connu pour induire des néovascularisations choroïdiennes chez la souris suite à l'augmentation du VEGF. Ce vecteur a ensuite été utilisé dans le modèle de dégénération induite par la lumière. Les résultats des électrorétinogrammes, les mesures de l'acuité visuelle et les analyses histologiques ont montré que l'injection du LV-V65 contribue à la maintenance de photorécepteurs fonctionnels. Au niveau de l'EP, l'absence d'albumine et la maintenance des jonctions cellulaires des cellules de l'EP ont démontré que l'intégrité de la barrière hémato-rétinienne externe est préservée suite au traitement.Par conséquent, tous les résultats obtenus indiquent que le VEGF est un médiateur important impliquée dans le processus de dégénération induit par la lumière et confirme le rôle cruciale de la balance entre les facteurs pro- et anti-angiogéniques dans la survie des photorécepteurs. Cette étude révèle également l'importance de l'intégrité de la barrière hémato-rétinienne et l'importance du lien fonctionnel et structurel entre l'EP et les photorécepteurs, essentiel pour la survie de ces derniers. Par ailleurs, Cette étude suggère que des dérèglements au niveau de l'équilibre du VEGF ne sont pas seulement impliqués dans la forme humide de la DMLA, comme déjà démontré dans des études antérieures, mais pourraient également contribuer et survenir dans des formes précoces de la DMLA, et par conséquent le VEGF représente une cible thérapeutique potentielle pour les maladies associées à des anomalies au niveau de l'EP.

Antimicrobial Activity of Iberian macroalgae

The antibacterial and antifungal activity of 82 marine macroalgae (18 Chlorophyceae, 25 Phaeophyceae and 39 Rhodophyceae) was studied to evaluate their potential for being used as natural preservatives in the cosmetic industry. The bioactivity was analysed from crude extracts of fresh and lyophilised samples against three Gram-positive bacteria, two Gram-negative bacteria and one yeast using the agar diffusion technique. The samples were collected seasonally from Mediterranean and Atlantic coasts of the Iberian Peninsula. Of the macroalgae analysed, 67% were active against at least one of the six test microorganisms. The highest percentage of active taxa was found in Phaeophyceae (84%), followed by Rhodophyceae (67%) and Chlorophyceae (44%). Nevertheless, red algae had both the highest values and the broadest spectrum of bioactivity. In particular, Bonnemaisonia asparagoides, Bonnemaisonia hamifera, Asparagopsis armata and Falkenbergia rufolanosa (Bonnemaisoniales) were the most active taxa. Bacillus cereus was the most sensitive test microorganism and Pseudomonas aeruginosa was the most resistant. The highest percentages of active taxa from Phaeophyceae and Rhodophyceae were found in autumn, whereas they were found in summer for Chlorophyceae.

Virtual screening and computational optimization for the discovery of covalent prolyl oligopeptidase inhibitors with activity in human cells.

Our docking program, Fitted, implemented in our computational platform, Forecaster, has been modified to carry out automated virtual screening of covalent inhibitors. With this modified version of the program, virtual screening and further docking-based optimization of a selected hit led to the identification of potential covalent reversible inhibitors of prolyl oligopeptidase activity. After visual inspection, a virtual hit molecule together with four analogues were selected for synthesis and made in one-five chemical steps. Biological evaluations on recombinant POP and FAPα enzymes, cell extracts, and living cells demonstrated high potency and selectivity for POP over FAPα and DPPIV. Three compounds even exhibited high nanomolar inhibitory activities in intact living human cells and acceptable metabolic stability. This small set of molecules also demonstrated that covalent binding and/or geometrical constraints to the ligand/protein complex may lead to an increase in bioactivity.

Comprehensive approach for the detection of antifungal compounds using a susceptible strain of Candida albicans and confirmation of in vivo activity with the Galleria mellonella model.

An efficient screening strategy for the identification of potentially interesting low-abundance antifungal natural products in crude extracts that combines both a sensitive bioautography assay and high performance liquid chromatography (HPLC) microfractionation was developed. This method relies on high performance thin layer chromatography (HPTLC) bioautography with a hypersusceptible engineered strain of Candida albicans (DSY2621) for bioactivity detection, followed by the evaluation of wild type strains in standard microdilution antifungal assays. Active extracts were microfractionated by HPLC in 96-well plates, and the fractions were subsequently submitted to the bioassay. This procedure enabled precise localisation of the antifungal compounds directly in the HPLC chromatograms of the crude extracts. HPLC-PDA-mass spectrometry (MS) data obtained in parallel to the HPLC antifungal profiles provided a first chemical screening about the bioactive constituents. Transposition of the HPLC analytical conditions to medium-pressure liquid chromatography (MPLC) allowed the efficient isolation of the active constituents in mg amounts for structure confirmation and more extensive characterisation of their biological activities. The antifungal properties of the isolated natural products were evaluated by their minimum inhibitory concentration (MIC) in a dilution assay against both wild type and engineered strains of C. albicans. The biological activity of the most promising agents was further evaluated in vitro by electron microscopy and in vivo in a Galleria mellonella model of C. albicans infection. The overall procedure represents a rational and comprehensive means of evaluating antifungal activity from various perspectives for the selection of initial hits that can be explored in more in-depth mode-of-action studies. This strategy is illustrated by the identification and bioactivity evaluation of a series of antifungal compounds from the methanolic extract of a Rubiaceae plant, Morinda tomentosa, which was used as a model in these studies.

Plant compounds insecticide activity against Coleoptera pests of stored products

The objective of this work was to screen plants with insecticide activity, in order to isolate, identify and assess the bioactivity of insecticide compounds present in these plants, against Coleoptera pests of stored products: Oryzaephilus surinamensis L. (Silvanidae), Rhyzopertha dominica F. (Bostrichidae) and Sitophilus zeamais Mots. (Curculionidae). The plant species used were: basil (Ocimum selloi Benth.), rue (Ruta graveolens L.), lion's ear (Leonotis nepetifolia (L.) R.Br.), jimson weed (Datura stramonium L.), baleeira herb (Cordia verbenacea L.), mint (Mentha piperita L.), wild balsam apple (Mormodica charantia L.), and billy goat weed or mentrasto (Ageratum conyzoides L.). The insecticide activity of hexane and ethanol extracts from those plants on R. dominica was evaluated. Among them, only hexane extract of A. conyzoides showed insecticide activity; the hexane extract of this species was successively fractionated by silica gel column chromatography, for isolation and purification of the active compounds. Compounds 5,6,7,8,3',4',5'-heptamethoxyflavone; 5,6,7,8,3'-pentamethoxy-4',5'-methilenedioxyflavone and coumarin were identified. However, only coumarin showed insecticide activity against three insect pests (LD50 from 2.72 to 39.71 mg g-1 a.i.). The increasing order of insects susceptibility to coumarin was R. dominica, S. zeamais and O. surinamensis.