The use of enzyme catalysts in organic media for the synthesis of biodegradable polyesters

The enzyme catalysed polytransesterification of diesters with diols was investigated under various conditions. The most consistent results were obtained using crude porcine pancreatic lipase (PPL) suspended in anhydrous diethyl ether. Addition of molecular sieve to the above system gave higher molecular weight products. The PPL catalysed reaction of bis(2,2,2-trichlorethyl) adipate and glutarate with butane-1,4-diol in anhydrous ether with and without molecular sieve was investigated over a range of times from 8 to 240 hours. The 72 hour adipate reaction with molecular sieve gave the highest molecular weight polymer (Mn 6,500 and Mw 9,400). The glutarate gave the maximum molecular weight polyester after 24 hours (Mn 5,700 and Mw 9,500). Occasionally the glutarate reaction produced very high molecular weight polyester-enzyme complexes. Toluene generally gave lower molecular weight products than diethyl ether. Dichloromethane and tetrahydrofuran gave mainly dimers and trimers. Alternative enzyme and diol systems were also investigated. These yielded no polymeric products. The molecular weights of the polyesters were determined by 1H NMR end-group analysis and by GPC. The molecular weights determined by NMR were on average about twice as great as those determined by GPC. The synthesis of the following diesters is described: i)Bis(2,2,2-trichloroethyl) succinate, glutarate, adipate, trans-3-hexenedioate, and trans-3,4-epoxyadipate. ii) Diphenyl glutarate and adipate.iii)Bis(2,2,2-fluoroethyl) glutarate and trans-3-hexendioate.iv) Divinyl glutarate. v) N,N'Glutaryl dicyclohexanone oxime.The polytransesterification of all the above esters with diols was investigated. The easily synthesised bis(2,2,2-trichloroethyl) glutarate and adipate gave the best results and the work was concentrated on these two esters.

Uptake and transport of orally-deliverable drugs across caco-2 cell monolayers:the effect of lipid formulations

The aim of this thesis is to investigate the physicochemical parameters which can influence drug loading within liposomes and to characterise the effect such formulations have on drug uptake and transport across in vitro epithelial barrier models. Liposomes composed of phosphatidylcholine (PC) or distearoyl phosphatidylcholine (DSPC) and cholesterol (0, 4, 8, 16 µM) were prepared and optimised in terms of drug loading using the hand-shaking method (Bangham et al., 1965). Subsequently, liposomes composed of 16 µM PC or DSPC and cholesterol (4 µM) were used to monitor hydroxybenzoate release and transport from Iiposomes. The MIT (3[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and crystal violet assays were employed to determine toxicity of the Iiposome. formulations towards the Caco-2 cell line, employed to model the epithelial barrier in vitro. Uptake and transport of mannitol, propranolol, glutamine and digoxin was measured in the presence and absence of Iiposome formulations to establish changes in absorption resulting from the presence of lipid formulations. Incorporation of the four hydroxybenzoates was shown to be influenced by a number of factors, including liposome composition and drug conformation. Methyl hydroxybenzo.ate (MP) was incorporated into the bilayer most effectively with percentage incorporation of 68% compared to 45% for butyl hydroxybenzoate (BP), despite its increased Iipophilicity. This was attributed to the decreased packing ability of BP within the hydrocarbon core of the lipid bilayer compared to MP. Release studies also suggested that the smaller MP was more strongly incorporated within the lipid bilayer with only 8% of the incorporated solute being released after 48-hours compared to 17% in the case of BP. Model transport studies were seen to reflect drug release profiles from the liposome bilayers with significantly (p < 0.01) higher amounts of BP partitioning from the liposome compared to MP, Caco-2 cell viability was maintained above 86% in the presence of all Iiposome formulations tested indicating the liposome formulations are non-toxic towards Caco-2 cells. Paracellular (apical-to-basolateral) transport of mannitol was significantly increased in the presence of DSPC, PC / DSPC:Cholesterol (16:4 µM; 1000 µg). Glutamine uptake and transport via the carrier-mediated route was Significantly (p < 0.01) increased in the presence of PC I DSPC:Cholesterol (16:0; 16:4 µM). Digoxin apical-to-basolateral transport was significantly increased (p < 0,01) in the presence of PC / DSPC:Cholesterol (16:0; 16:4 µM); thus reducing digoxin efflux via P-glycoprotein. In contrast, PC:ChoJesterol (16:0; 16:4 µM) significantly (p < 0.01) decreased propranolol uptake via the passive transcellular route. Bi-directional transport of propranolol was significantly (p < 0,01) decreased in the presence of PC/DSPC:Cholesterol (16:0; 16:4 µM). The structure of a solute is an important determinant for the incorporation and release of a solute from liposome formulations. PC, DSPC and cholesterol liposome formulations are nontoxic towards Caco-2 cell monolayers and improved uptake and transport of mannitol, glutamine. and digoxin across Caco-2 cell monolayers; thus providing a potential alternative delivery vehicle.

Synthesis and characterization of triazolotriazines

The Dimroth rearrangement in ring-fused 1,2,4-triazoles has been reviewed in detail in Part I and the synthesis of all known triazolo-triazines is described in Part II. Experimental investigations concerned the establishinent of the skeletal arrangement of a variety of triazolotriazines formed by several synthetic routes. Interaction of 3-amino-5-hydrazino-12,4-triazole and benzilafforded 2-amino-6, 7-diphenyl-1, 2,4-triazolo[ 5, 1-c-]-1,2,4-triazine,whereas cyclization of 5,6-diphenyl-3-hydrazino-1,2,4-triazine withcyanogen bromide resulted in the isomeric 3-amino-6,7-diphenyl,-1,2,4-triazolo [4, 3-b]-1,2,4-triazine: both amines were deaminated with amyl nitrite in boiling tetrahydrofuran without rearrangement of the heterocyclic skeleton. 6,7-Diphenyl-1,2,4-triazolo[5,1-cJ-1,2.4-triazine, synthesized from 3-hydrazino-1,2,4-triazole and benzil, formed a covalent hydrate which could be detected spectroscopically in solution, and a covalemt methanolate and ethanolate which could be isolated. A new route to 3-amino-5-hydrazino-pyrazole is described and cyclization to 7-amino-3,4-diphenyl-pyrazolo[ 5,1-.c]-1,2,4-triazine was achieved with benzil. The diazonium nitrate of 3-amino-1,2,4-triazole coupled with ethyl cyanoacetate to yield a mixture of two geometrical isomers of ethyl 2-(2H-1,2,4-triazol-3-ylhydrazono) cyanoacetate.Recrystallization of the crude coupling mixture from aqueous ethanol gave a single hydra-zone which cyclized predominantly to ethyl 7-amino-1,2,4-triazolo[5,1-c]-1,2,4-triazine-6-carboxylate in acid conditions and 6-cyano-1,2,4-triazolo[ 5,1-c]-1,2,4~triazin-7(4H)-one under basic conditions. The nature of the cyclizing medium also controlled the cyclization of .the (pyrazol-ylhydrazono) cyanoacetate hut the corresponding (tetrazol- ylhydrazono) cyanoacetate gave only ethyl 7-aminotetrazolo[ 5,1-cJ-1,2,4- -triazine-6-carboxylate. 2-( 2H-1,2,4-Triazol-3-:ylhydrazono) malonitrile cyclized unambiguously to 7-amino-6-cyano-1,2,4-triazolo-[ 5,1-c]-1,2,4- triazine. Drastic hydrolysis of ethyl 2-(2H-1,2,4-triazol-3-yllhydrazono)-cyanoacetate, ethyl 7-amino-1, 2,4-triazolo[ 5,1-c]-1,2,4-triazine-6-carboxylate, 6-cyano-1,2,4-triazolo[ 5,1-c]-1,2,4-triazin-7{ 4H)-one and 7-amino-6- cyano-1,2,4-triazolo[5,1-c]-1,2,4-triazine gave a hydrate of 1,2,4-triazo1o[5,1-c ]-1,2,4-triazin-7(4H)-one. Mass spectral fragmentations of 7-aminoazolo-[5,1-c]-1,2,4-triazinesconfirm that the azole ring is more stable than the 1,2,4-triazine ring on electron impact.

Free radicals as potential antitumour agents

The aim of this work was to use extremely low concentrations of free radical generating compounds as a 'catalyst' to trigger endogenous free radical chain reactions in the host and to selectively eliminate neoplastic cells in the host. To test the hypothesis, a number of free radical generating compounds were screened on several malignant cell lines in vitro to select model compounds that were used against tumour models in vivo. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and its derivatives were selected at the model compounds for in vivo experiments in view of their high cytotoxic potency against several malignant cell lines in vitro. The water soluble derivative, 2,2-diphenyl-1-(2', 4'-dinitro-6'-sulphophenyl) hydrazyl (DDSH) given by subcutaneous injections demonstrated significant antitumour activities against the MAC 16 murine colon adenocarcinoma implanted subcutaneously in male NMRI mice at nanomolar concentration range. 40-60% of long term survival of over 60 days was achieved (compared with control survival of 20 days) with total tumour elimination. This compound was also active against both P388 leukaemia in male BDF1 mice and TLX5 lymphoid tumour in male CBA/CA mice at a similar concentration range. However, some of these animals died suddenly after treatment with no evidence of disease present at post mortem. The cause of death was unknown but thought to be related to the treatment. There was significant increase in serum level of malondialdehyde (MDA) following treatment, but did not correlate to the antitumour activities of these compounds. Induction of supcroxide dismutase (SOD), and glutathione peroxidase (GPx) occurred around day 8 after the administration of DDSH. Histological sections of MAC16 tumours showed areas of extensive massive haemorrhagic necrosis and vascular collapse associated with perivascular cell death following the administration of nanomolar concentration of DDSH which was probably compatible with the effects of free radicals. It was concluded that the antitumour activities of these compounds may be related to free radical and cytokine production.

Synthesis and evaluation of N-(3-oxo-2,3-dihydro-1H-pyrazol-4-yl)-1H-indole-carboxamides as cholecystokinin antagonists

The structure-activity relationship optimization of the pyrazoline template 3a resulted in novel 3-oxo-1,2-diphenyl-2,3-dihydro-1H-pyrazol-4-yl)-indole carboxamides 4a-4e. These non-peptidal CCK ligands have been shown to act as potent CCK 1 ligands in a [125]I-CCK-8 receptor binding assay. The best amides (4c and 4d) of this series displayed an IC50 of 20/25 CCK 1 for the CCK 1 receptor. In a subsequent in-vivo evaluation using various behaviour pharmacological assays, an anxiolytic effect of these novel 3-oxo-1,2-diphenyl-2,3-dihydro-1H-pyrazol-4-yl)-indole carboxamides was found at high doses in the elevated plus-maze. In the despair swimming test, a model for testing antidepressants, an ED50 of 0.33/0.41 mg kg -1 was determined for amide 4c/4d and the antidepressant effect had a magnitude comparable to desimipramine. © 2006 The Authors.

Competitive processes in controlled cationic ring-opening polymerization of oxetane:a Lotka-Volterra predator-prey model of two growing species competing for the same resources

The activation-deactivation pseudo-equilibrium coefficient Qt and constant K0 (=Qt x PaT1,t = ([A1]x[Ox])/([T1]x[T])) as well as the factor of activation (PaT1,t) and rate constants of elementary steps reactions that govern the increase of Mn with conversion in controlled cationic ring-opening polymerization of oxetane (Ox) in 1,4-dioxane (1,4-D) and in tetrahydropyran (THP) (i.e. cyclic ethers which have no homopolymerizability (T)) were determined using terminal-model kinetics. We show analytically that the dynamic behavior of the two growing species (A1 and T1) competing for the same resources (Ox and T) follows a Lotka-Volterra model of predator-prey interactions. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

New genre of antioxidants from renewable natural resources:synthesis and characterisation of rosemary plant-derived antioxidants and their performance in polyolefins

Several ester derivatives of rosmarinic acid (rosmarinates) were synthesised, characterised (1D and 2D NMR, UV and FTIR spectroscopy) and tested for their potential use as antioxidants derived from a renewable natural resource. The intrinsic free radical scavenging activity of the rosmarinates was assessed, initially using a modified DPPH (2, 2-diphenyl-1-picrylhydrazyl radical) method, and found to be higher than that of commercial synthetic hindered phenol antioxidants Irganox 1076 and Irganox 1010. The thermal stabilising performance of the rosmarinates in polyethylene (PE) and polypropylene (PP) was subsequently examined and compared to that of samples prepared similarly but in the presence of Irganox 1076 (in PE) and Irganox 1010 (in PP) which are typically used for polyolefin stabilisation in industrial practice. The melt stability and the long-term thermo-oxidative stability (LTTS) of processed polymers containing the antioxidants were assessed by measuring the melt flow index (MFI), melt viscosity, oxidation induction time (OIT) and long-term (accelerated) thermal ageing performance. The results show that both the melt and the thermo-oxidative stabilisation afforded by the rosmarinates, and in particular the stearyl derivative, in both PE and PP, are superior to those of Irganox 1076 and Irganox 1010, hence their potential as effective sustainable bio-based antioxidants for polymers. The rosmarinic acid used for the synthesis of the rosmarinates esters in this study was obtained from commercial rosemary extracts (AquaROX80). Furthermore, a large number of different strains of UK-grown rosemary plants (Rosmarinum officinalis) were also extracted and analysed in order to examine their antioxidant content. It was found that the carnosic and the rosmarinic acids, and to a much lesser extent the carnosol, constituted the main antioxidant components of the UK-plants, with the two acids being present at a ratio of 3:1, respectively.

Extração, caracterização e avaliação bioativa do extrato de Arrabidaea chica

The use of plants for medicinal purposes is ancient, with widespread application in medicinal drugs. Although plants are promising sources for the discovery of new molecules of pharmacological interest, estimates show that only 17% of them have been studied for their possible use in medicine. Thus, biodiversity of Brazilian flora represents an immense potential for economic use by the pharmaceutical industry. The plant Arrabidaea chica, popularly known as “pariri”, is common in the Amazon region, and it is assigned several medicinal properties. The leaves of this plant are rich in anthocyanins, which are phenolic compounds with high antioxidant power. Antioxidant compounds play a vital role in the prevention of neurological and cardiovascular diseases, cancer and diabetes, among others. Within the anthocyanins found in Arrabidaea chica, stands out Carajurin (6,7-dihydroxy-5,4’- dimethoxy-flavilium), which is the major pigment encountered in this plant. The present work aimed to study on supercritical extraction and conventional extraction (solid-liquid extraction) in leaves of Arrabidaea chica, evaluating the efficiency of the extractive processes, antioxidant activity and quantification of Carajurin contained in the extracts. Supercritical extraction used CO2 as solvent with addition of co-solvent (ethanol/water mixture) and were conducted by the dynamic method in a fixed bed extractor. The trials followed a 24-1 fractional factorial design, the dependent variables were: process yield, concentration of Carajurin and antioxidant activity; and independent variables were: pressure, temperature, concentration of co-solvent (v/v) and concentration of water in the co-solvent mixture (v/v). Yields (mass of dry extract/mass of raw material used) obtained from supercritical extraction ranged from 15.1% to 32%, and the best result was obtained at 250 bar and 40 °C, co-solvent concentration equal to 30% and concentration of water in the co-solvent mixture equal to 50%. Through statistical analysis, it was found that the concentration of co-solvent revealed significant effect on the yield. Yields obtained from conventional extractions were of 8.1% (water) and 5.5% (ethanol). Through HPLC (High-performance liquid chromatography) analysis, Carajurin was quantified in all the extracts and concentration values (Carajurin mass/mass of dry extract) ranged between 1% and 2.21% for supercritical extraction. For conventional extraction, Carajurin was not detected in the aqueous extract, while the ethanol extract showed Carajurin content of 7.04%, and therefore, more selective in Carajurin than the supercritical extraction. Evaluation of antioxidant power (radical 2,2-diphenyl-1-picrylhydrazyl – DPPH – sequestration method) of the supercritical extracts resulted in EC50 values (effective concentration which neutralizes 50% of free radicals) ranged from 38.34 to 86.13 μg/mL, while conventional extraction resulted in EC50 values of 167.34 (water) and 42.58 (ethanol) μg/mL. As for the quantification of total phenolic content (Folin-Ciocalteau analysis) of the supercritical extracts resulted in values ranged from 48.93 and 88.62 mg GAE/g extract (GAE = Gallic Acid Equivalents), while solid-liquid extraction resulted in values of 37.63 (water) and 80.54 (ethanol) mg GAE/g extract. The good antioxidant activity cannot be attributed solely to the presence of Carajurin, but also the existence of other compounds and antioxidants in Arrabidaea chica. By optimizing the experimental design, it was possible to identify the experiment that presented the best result considering the four dependent variables together. This experiment was performed under the following conditions: pressure of 200 bar, temperature of 40 °C, co-solvent concentration equal to 30% and concentration of water in the co-solvent mixture equal to 30%. It is concluded that, within the studied range, it is possible to purchase the optimum result using milder operating conditions, which implies lower costs and greater ease of operation.

Estudo fitoquímico e biológico das folhas de Banisteriopsis argyrophylla (A. Juss.) B. Gates (Malpighiaceae)

With the increasing fungi resistance compared with existing drugs on the market and the side effects reported by some compounds with antioxidant properties and enzymatic inhibitors, in particular against α-amylase and α-glucosidase, the discovery of new compounds with biological potential, becomes a need. In this context, natural products can be an important source for the discovery of new active molecular architectures. Then, this study aimed to evaluate the antioxidant activity, the enzymatic inhibitory activity of α-amylase and α-glucosidase, the antifungal and cytotoxic activities of ethanolic extract (EE) the leaves of Banisteriopsis argyrophylla (Malpighiaceae) and their fractions, obtained by liquid-liquid extraction using solvents of increasing polarity. The antioxidant activity was evaluated by the free radical DPPH scavenging method (2,2-diphenyl-1-picrylhydrazyl) and the ethyl acetate fractions (FAE) and n-butanol (FB) were the most active, confirmed by the peak current and the oxidation potential obtained by differential pulse voltammetry (DPV). The inhibitory activity of the α-amylase and α-glucosidase was analyzed considering the reactions between substrates α-(2-chloro-4-nitrophenyl)-β-1,4-galactopiranosilmaltoside (Gal-α-G2-CNP) and 4-nitrophenyl-α-D-glucopyranoside (p-NPG), respectively. Initially, it was found that the EE showed considerable activity against α-amylase (EC50 = 2.89±0.1 μg m L–1) compared to the acarbose used as positive control (EC50 = 0.08±0.1 μg mL–1) and that did not showed promising activity against the α-glucosidase. After this observation we evaluated the inhibitory activity of α-amylase fractions, with FAE (EC50 = 2.33±0.1 μg mL–1) and FB (EC50 = 2.57 ± 0.1 μg mL–1) showing the best inhibitions. The antifungal activity was evaluated against Candida species, and the FAE had better antifungal potential (MIC's between 93.75 and 11.72 μg mL–1) compared with amphotericin as positive standard (MIC = 1.00 and 2.00 μg L–1 for C. parapsilosis and C. krusei used as controls, respectively). The EE (CC50 = 360.00 ± 12 μg mL–1) and fractions (CC50's> 270.00 μg mL–1) were considerably less toxic to Vero cells than the cisplatin used as positive control (CC50 = 7.01 ± 0 6 μg mL–1). The FAE showed the best results for the activities studied, this fraction was submitted to ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS)), and the following flavonoids have been identified: (±)-catechin, quercetin-3-O-β-D-Glc/ quercetin-3-O-β-D-Gal, quercetin-3-O-β-L-Ara, quercetin-3-O-β-D-Xyl, quercetin-3-O-α-L-Rha, kaempferol-3-O-α-L-Rha, quercetin-3-O-(2''-galoil)-α-L-Rha, quercetin-3-O-(3''-galoil)-α-L-Rha and kaempferol-3-O-(3''-galoil)-α-L-Rha,. FAE was submitted to column chromatography using C18 phase, and (±)-catechin was isolated (FAE-A1, 73 mg) and three fractions consisting of a mixture of flavonoids were obtained (FAE-A2, FAE-A3 and FAE-A4). These compounds were identified by thin layer chromatography (TLC) and (–)-ESI-MS. The (±)-catechin fraction showed an MIC = 2.83 μg ml–1 in assay using C. glabrata, with amphotericin as positive control. The fractions FAE-A2, FAE-A3, FAE-A4, showed less antifungal potential in tested concentrations. The identified flavonoids are described in the literature, regarding their antioxidant capacity and (±)-catechin, quercetin-3-O-Rha and kaempferol-3-O-Rha are described as α-amylase inhibitors. Thus, B. argyrophylla is an important species that produces compounds with antioxidant potential that can be related to the traditional use as anti-inflammatory and also has antifungal compounds and inhibitors of α-amylase. Therefore, these leaves are promising resources for the production of new drugs.

Avaliação da sensibilidade ao H2O2 de diferentes linhas celulares humanas: estabelecimento de modelos celulares para avaliação da capacidade antioxidante de moléculas ou extratos

Os organismos marinhos são considerados uma fonte de novos compostos bioativos com enorme potencial biotecnológico. As bactérias associadas a macroalgas têm vindo a ser estudadas devido à produção de metabolitos secundários com atividades biológicas. Neste trabalho foram isoladas e identificadas 90 bactérias associadas às macroalgas Asparagopsis armata, Bifurcaria bifurcata e Sphaerococcus coronopifolius com diferentes características fenotípicas, sendo identificadas relativamente ao seu género através da sequenciação do gene 16S RNA. A extração de compostos bioativos foi realizada com os solventes metanol e diclorometano (1:1). A capacidade antioxidante dos extratos das bactérias associadas foi avaliada através do método fluorimétrico ORAC (oxygen radical absorbent capacity), da quantificação total de polifenóis (QTP) e da capacidade de redução do radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). O efeito citotóxico do H2O2 foi testado nos modelos celulares SH-SY5Y, MCF-7 e HepG-2, representativos de células humanas neuronais, epiteliais da glândula mamária e hepáticas, respetivamente. Os extratos com maior capacidade antioxidante foram testados nos modelos celulares em condições de stress oxidativo induzido pelo H2O2. Os resultados foram revelados pelo método de 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT). O género de bactérias mais representativo identificado em associação com Asparagopsis armata, Bifurcaria Bifurcata e Sphaerococcus coronopifolius foi Vibrio sp. com 40%, 48,72% e 28,57%, respetivamente. Os géneros de bactérias menos representativos identificados em associação com Asparagopsis armata foram Bacillus sp., Cobetia sp. e Erwinia sp., com uma ocorrência de 3,33%. Por sua vez, Citricoccus sp., Cellulophaga sp., Ruegeria sp. e Staphylococcus sp. foram os géneros de bactérias menos representativos associados a Bifurcaria Bifurcata (2,56%). Os géneros menos representativos identificados em associação com Sphaerococcus coronopifolius foram Bacillus sp. e Holomonas sp. com uma ocorrência de 9,52%. O extrato da bactéria associada que apresentou maior potencial antioxidante avaliado pelos métodos de ORAC (3603,66 ± 80,14 μmol eq. Trolox/g extrato), QTP (53,854 ± 3,02 mg eq. ácido gál./g extrato) e DPPH (20,21 (14,41-28,34) μg.mL-1) foi a BB16 (Shewanella sp.), associada à alga Bifurcaria bifurcata. O efeito induzido pelo H2O2 foi bastante distinto na redução da viabilidade celular, com IC50 distintos, nas células SH-SY5Y (206,0 μM (150,4 – 282,2)), MCF-7 (450,2 μM (388,0 – 522,5)) e HepG-2 (1058,0 μM (847,3 – 1321,0)).A elevada atividade antioxidante do extrato da bactéria associada à alga Bifurcaria bifurcata (0,1mg.mL-1; BB16 – Shewanella sp.) permitiu a prevenção do efeito induzido pelo H2O2 na linha celular SH-SY5Y (IC50 - 431,7 μM (360,1 – 517,6). Em conclusão, as bactérias associadas das macroalgas Asparagopsis armata, Bifurcaria bifurcata e Sphaerococcus coronopifolius podem ser uma excelente e interessante fonte de compostos marinhos naturais com um elevado potencial antioxidante.

Microbialites and lipid biomarker from coral reefs off the volcanic islands Tahiti and Vanuatu and from the nonvolcanic sites Belize and the Maldives

The occurrence of microbialites in post-glacial coral reefs has been interpreted to reflect an ecosystem response to environmental change. The greater thickness of microbialites in reefs with a volcanic hinterland compared to thinner microbial crusts in reefs with a non-volcanic hinterland led to the suggestion that fertilization of the reefal environment by chemical weathering of volcanic rocks stimulated primary productivity and microbialite formation. Using a molecular and isotopic approach on reef-microbialites from Tahiti (Pacific Ocean), it was recently shown that sulfate-reducing bacteria favored the formation of microbial carbonates. To test if similar mechanisms induced microbialite formation in other reefs as well, the Tahitian microbialites are compared with similar microbialites from coral reefs off Vanuatu (Pacific Ocean), Belize (Caribbean Sea, Atlantic Ocean), and the Maldives (Indian Ocean) in this study. The selected study sites cover a wide range of geological settings, reflecting variable input and composition of detritus. The new lipid biomarker data and stable sulfur isotope results confirm that sulfate-reducing bacteria played an intrinsic role in the precipitation of microbial carbonate at all study sites, irrespective of the geological setting. Abundant biomarkers indicative of sulfate reducers include a variety of terminally-branched and mid chain-branched fatty acids as well as mono-O-alkyl glycerol ethers. Isotope evidence for bacterial sulfate reduction is represented by low d34S values of pyrite (-43 to -42 per mill) enclosed in the microbialites and, compared to seawater sulfate, slightly elevated d34S and d18O values of carbonate-associated sulfate (21.9 to 22.2 per mill and 11.3 to 12.4 per mill, respectively). Microbialite formation took place in anoxic micro-environments, which presumably developed through the fertilization of the reef environment and the resultant accumulation of organic matter including bacterial extracellular polymeric substances (EPS), coral mucus, and marine snow in cavities within the coral framework. ToF-SIMS analysis reveals that the dark layers of laminated microbialites are enriched in carbohydrates, which are common constituents of EPS and coral mucus. These results support the hypothesis that bacterial degradation of EPS and coral mucus within microbial mats favored carbonate precipitation. Because reefal microbialites formed by similar processes in very different geological settings, this comparative study suggests that a volcanic hinterland is not required for microbialite growth. Yet, detrital input derived from the weathering of volcanic rocks appears to be a natural fertilizer, being conductive for the growth of microbial mats, which fosters the development of particularly abundant and thick microbial crusts.

(Tables S1,2) Stable isotopes in muscle tissue of polar bears (Ursus maritimus) from various regions of the Arctic

The relative contribution of regional contamination versus dietary differences to geographic variation in polar bear (Ursus maritimus) contaminant levels is unknown. Dietary variation between Alaska, Canada, East Greenland, and Svalbard subpopulations was assessed by muscle nitrogen and carbon stable isotope (d15N, d13C) and adipose fatty acid (FA) signatures relative to their main prey (ringed seals). Western and southern Hudson Bay signatures were characterized by depleted d15N and d13C, lower proportions of C20 and C22 monounsaturated FAs and higher proportions of C18 and longer chain polyunsaturated FAs. East Greenland and Svalbard signatures were reversed relative to Hudson Bay. Alaskan and Canadian Arctic signatures were intermediate. Between-subpopulation dietary differences predominated over interannual, seasonal, sex, or age variation. Among various brominated and chlorinated contaminants, diet signatures significantly explained variation in adipose levels of polybrominated diphenyl ether (PBDE) flame retardants (14-15%) and legacy PCBs (18-21%). However, dietary influence was contaminant class-specific, since only low or nonsignificant proportions of variation in organochlorine pesticide (e.g., chlordane) levels were explained by diet. Hudson Bay diet signatures were associated with lower PCB and PBDE levels, whereas East Greenland and Svalbard signatures were associated with higher levels. Understanding diet/food web factors is important to accurately interpret contaminant trends, particularly in a changing Arctic.

A Selective Irreversible Inhibitor of Furin Does Not Prevent Pseudomonas Aeruginosa Exotoxin A-Induced Airway Epithelial Cytotoxicity

Many bacterial and viral pathogens (or their toxins), including Pseudomonas aeruginosa exotoxin A, require processing by host pro-protein convertases such as furin to cause dis- ease. We report the development of a novel irreversible inhibitor of furin (QUB-F1) consist- ing of a diphenyl phosphonate electrophilic warhead coupled with a substrate-like peptide (RVKR), that also includes a biotin tag, to facilitate activity-based profiling/visualisation. QUB-F1 displays greater selectivity for furin, in comparison to a widely used exemplar com- pound (furin I) which has a chloromethylketone warhead coupled to RVKR, when tested against the serine trypsin-like proteases (trypsin, prostasin and matriptase), factor Xa and the cysteine protease cathepsin B. We demonstrate QUB-F1 does not prevent P. aerugi- nosa exotoxin A-induced airway epithelial cell toxicity; in contrast to furin I, despite inhibiting cell surface furin-like activity to a similar degree. This finding indicates additional proteases, which are sensitive to the more broad-spectrum furin I compound, may be involved in this process.

Chemical characterization and evaluation of biological activity of Cynara cardunculus extractable compounds

The Mediterranean species Cynara cardunculus L. is recognized in the traditional medicine, for their hepatoprotective and choleretic effects. Biomass of C. cardunculus L. var. altilis (DC), or cultivated cardoon, may be explored not only for the production of energy and pulp fibers, but also for the extraction of bioactive compounds. The chemical characterization of extractable components, namely terpenic and phenolic compounds, may valorize the cultivated cardoon plantation, due to their antioxidant, antitumoral and antimicrobial activities. In this study, the chemical composition of lipophilic and phenolic fractions of C. cardunculus L. var. altilis (DC), cultivated in the south of Portugal (Baixo Alentejo region) was characterized in detail, intending the integral valorization of its biomass. The biological activity of cultivated cardoon extracts was evaluated in terms of antioxidant, human tumor cell antiproliferative and antibacterial effects. Gas chromatography-mass spectrometry (GC-MS) was used for the chemical analysis of lipophilic compounds. Sixty-five lipophilic compounds were identified, from which 1 sesquiterpene lactone and 4 pentacyclic triterpenes were described, for the first time, as cultivated cardoon components, such as: deacylcynaropicrin, acetates of β- and α-amyrin, lupenyl acetate and ψ-taraxasteryl acetate. Sesquiterpene lactones were the major family of lipophilic components of leaves (≈94.5 g/kg), mostly represented by cynaropicrin (≈87.4 g/kg). Pentacyclic triterpenes were also detected, in considerably high contents, in the remaining parts of cultivated cardoon, especially in the florets (≈27.5 g/kg). Taraxasteryl acetate was the main pentacyclic triterpene (≈8.9 g/kg in florets). High pressure liquid chromatography-mass spectrometry (HPLC-MS) was utilized for the chemical analysis of phenolic compounds. Among the identified 28 phenolic compounds, eriodictyol hexoside was reported for the first time as C. cardunculus L. component, and 6 as cultivated cardoon components, namely 1,4-di-O-caffeoylquinic acid, naringenin 7-O-glucoside, naringenin rutinoside, naringenin, luteolin acetylhexoside and apigenin acetylhexoside. The highest content of the identified phenolic compounds was observed in the florets (≈12.6 g/kg). Stalks outer part contained the highest hydroxycinnamic acids abundance (≈10.3 g/kg), and florets presented the highest flavonoids content (≈10.3 g/kg). The antioxidant activity of phenolic fraction was examined through 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay. Stalks outer part, and receptacles and bracts extracts demonstrated the highest antioxidant effect on DPPH (IC50 of 34.35 μg/mL and 35.25 μg/mL, respectively). (cont.) abstract (cont.) The DPPH scavenging effect was linearly correlated with the total contents of hydroxycinnamic acids (r = -0.990). The in vitro antiproliferative activity of cultivated cardoon lipophilic and phenolic extracts was evaluated on a human tumor cells line of triple-negative breast cancer (MDA-MB-231), one of the most refractory human cancers to conventional therapeutics. After 48 h of exposition, leaves lipophilic extract showed higher inhibitory effect (IC50 = 10.39 μg/mL) than florets lipophilic extract (IC50 = 315.22 μg/mL), upon MDA-MB-231 cellular viability. Pure compound of cynaropicrin, representative of the main compound identified in leaves lipophilic extract, also prevented the cell proliferation of MDA-MB-231 (IC50 = 17.86 μM). MDA-MB-231 cells were much more resistant to the 48 h- treatment with phenolic extracts of stalks outer part (IC50 = 3341.20 μg/mL) and florets (IC50 > 4500 μg/mL), and also with the pure compound of 1,5-di-O-caffeoylquinic acid (IC50 = 1741.69 μM). MDA-MB-231 cells were exposed, for 48 h, to the respective IC50 concentrations of leaves lipophilic extract and pure compound of cynaropicrin, in order to understand their ability in modelling cellular responses, and consequently important potentially signaling pathways for the cellular viability decrease. Leaves lipophilic extract increased the caspase-3 enzymatic activity, contrarily to pure compound of cynaropicrin. Additionally, leaves lipophilic extract and pure compound of cynaropicrin caused G2 cell cycle arrest, possibly by upregulating the p21Waf1/Cip1 and the accumulation of phospho-Tyr15-CDK1 and cyclin B1. The inhibitory effects of leaves lipophilic extract and cynaropicrin pure compound, against the MDA-MB-231 cell proliferation, may also be related to the downregulation of phospho-Ser473-Akt. The antibacterial activity of cultivated cardoon lipophilic and phenolic extracts was assessed, for the first time, on two multidrug-resistant bacteria, such as the Gram-negative Pseudomonas aeruginosa PAO1 and the Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), two of the main bacteria responsible for health care-associated infections. Accordingly, the minimum inhibitory concentrations (MIC) were determined. Lipophilic and phenolic extracts of florets did not have antibacterial activity on P. aeruginosa PAO1 and MRSA (MIC > 2048 μg/mL). Leaves lipophilic extract did not prevent the P. aeruginosa PAO1 growth, but pure compound of cynaropicrin was slightly active (MIC = 2048 μg/mL). Leaves lipophilic extract and pure compound of cynaropicrin blocked MRSA growth (MIC of 1024 and 256 μg/mL, respectively). The scientific knowledge revealed in this thesis, either by the chemical viewpoint, or by the biological viewpoint, contributes for the valorization of C. cardunculus L. var. altilis (DC) biomass. Cultivated cardoon has potential to be exploited as source of bioactive compounds, in conciliation with other valorization pathways, and Portuguese traditional cheeses manufacturing.