Identificação de compostos que modulam a patogénese da doença de Machado-Joseph em C.elegans: autofagia como alvo terapêutico: identification of small compounds that modulate pathogenesis of Machado-Joseph disease in a C.elegans model: targeting autophagy

A doença de Machado-Joseph (DMJ) ou ataxia espinocerebelosa do tipo 3 (SCA3), conhecida por ser a mais comum das ataxias hereditárias dominantes em todo o mundo, é uma doença neurodegenerativa autossómica dominante que leva a uma grande incapacidade motora, embora sem alterar o intelecto, culminando com a morte do doente. Atualmente não existe nenhum tratamento eficaz para esta doença. A DMJ é resultado de uma alteração genética causada pela expansão de uma sequência poliglutamínica (poliQ), na região C-terminal do gene que codifica a proteína ataxina-3 (ATXN3). Os mecanismos celulares das doenças de poliglutaminas que provocam toxicidade, bem como a função da ATXN3, não são ainda totalmente conhecidos. Neste trabalho, usamos, pela sua simplicidade e potencial genético, um pequeno animal invertebrado, o nemátode C. elegans, com o objetivo de identificar fármacos eficazes para o combate contra a patogénese da DMJ, analisando simultaneamente o seu efeito na agregação da ATXN3 mutante nas células neuronais in vivo e o seu impacto no comportamento motor dos animais. Este pequeno invertebrado proporciona grandes vantagens no estudo dos efeitos tóxicos de proteínas poliQ nos neurónios, uma vez que a transparência das suas 959 células (das quais 302 são neurónios) facilita a deteção de proteínas fluorescentes in vivo. Para além disso, esta espécie tem um ciclo de vida curto, é económica e de fácil manutenção. Neste trabalho testámos no nosso modelo transgénico da DMJ com 130Qs em C.elegans dois compostos potencialmente moduladores da agregação da ATXN3 mutante e da resultante disfunção neurológica, atuando pela via da autofagia. De modo a validar a possível importância terapêutica da ativação da autofagia os compostos candidatos escolhidos foram o Litío e o análogo da Rapamicina CCI-779, testados independentemente e em combinação. A neuroproteção conferida pelo Litío e pelo CCI-779 independentemente sugere que o uso destes fármacos possa ser considerado uma boa estratégia como terapia para a DMJ, a testar em organismos evolutivamente mais próximos do humano. A manipulação da autofagia, segundo vários autores, parece ser benéfica e pode ser a chave para o desenvolvimento de novos tratamentos para várias doenças relacionadas com a agregação proteica e o envelhecimento.

Marine cyanobacteria compounds with anticancer properties: a review on the implication of apoptosis

Marine cyanobacteria have been considered a rich source of secondary metabolites with potential biotechnological applications, namely in the pharmacological field. Chemically diverse compounds were found to induce cytoxicity, anti-inflammatory and antibacterial activities. The potential of marine cyanobacteria as anticancer agents has however been the most explored and, besides cytotoxicity in tumor cell lines, several compounds have emerged as templates for the development of new anticancer drugs. The mechanisms implicated in the cytotoxicity of marine cyanobacteria compounds in tumor cell lines are still largely overlooked but several studies point to an implication in apoptosis. This association has been related to several apoptotic indicators such as cell cycle arrest, mitochondrial dysfunctions and oxidative damage, alterations in caspase cascade, alterations in specific proteins levels and alterations in the membrane sodium dynamics. In the present paper a compilation of the described marine cyanobacterial compounds with potential anticancer properties is presented and a review on the implication of apoptosis as the mechanism of cell death is discussed.

Antimicrobial activity of pyrazine and quinoxaline N,N’-dioxide heterocyclic compounds

The nitrogen heterocyclic organic compounds 1,4 dioxide pyrazine and quinoxaline derivatives have been widely studied due to their potential use as synthetic drugs. The thermochemical study of three N,N´-dioxides: 2,3,5-trimethylpyrazine-1,4-dioxide, tetramethylpyrazine-1,4-dioxide and 6-chloro-2,3-dimethilquinoxaline 1,4-dioxide has been recently developed in order to establish relationships among the energetical, structural and reactivity properties [4,5]. Several studies have reported their pharmacological activity, particularly as antimicrobial agents [1,2,3]. It has also been established a relation between energetical and structural properties and biological activity, once these compounds present N – oxide bonds, increasing their oxidative capacity. The present work reports the study of antimicrobial activity for those compounds against the bacteria Geobacillus stearothermophylus, Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli and also against the yeasts Saccharomyces cerevisiae PYCC 4072, Candida albicans PYCC3436T, Candida tropicalis PYCC, Issatchenka Orientalis PYCC. The determination of the minimal inhibitory concentration (MIC), points to an antimicrobial activity and the preliminary results indicate that these compounds may be potential candidates as antimicrobial drugs with clinical, agriculture or food industries applications.

Teas, dietary supplements and fruit juices: a comparative study regarding antioxidant activity and bioactive compounds

Nowadays, new emerging products claiming antioxidant properties are becoming more frequent. However, information about this topic in their labels is usually scarce. In this paper, we analyzed total phenolics, total flavonoids and ascorbic acid contents, as well as DPPH scavenging activity of several commercial samples, namely green tea and other herbal infusions, dietary supplements, and fruit juices, available in the Portuguese market. In general, beverages containing green tea and hibiscus showed higher phenolics contents (including flavonoids) and antioxidant activity than those without these ingredients. A borututu infusion presented the lowest concentrations of bioactive compounds and scavenging activity, due to the low recommended amount of plant to prepare the beverage. Some juices without antioxidant claims in the label presented similar values to those with it.

Espresso coffee residues: a valuable source of unextracted compounds

Espresso spent coffee grounds were chemically characterized to predict their potential, as a source of bioactive compounds, by comparison with the ones from the soluble coffee industry. Sampling included a total of 50 samples from 14 trademarks, collected in several coffee shops and prepared with distinct coffee machines. A high compositional variability was verified, particularly with regard to such water-soluble components as caffeine, total chlorogenic acids (CGA), and minerals, supported by strong positive correlations with total soluble solids retained. This is a direct consequence of the reduced extraction efficiency during espresso coffee preparation, leaving a significant pool of bioactivity retained in the extracted grounds. Besides the lipid (12.5%) and nitrogen (2.3%) contents, similar to those of industrial coffee residues, the CGA content (478.9 mg/100 g), for its antioxidant capacity, and its caffeine content (452.6 mg/100 g), due to its extensive use in the food and pharmaceutical industries, justify the selective assembly of this residue for subsequent use.

Optimization and validation of organochlorine compounds in adipose tissue by SPE-gas chromatography

Scientific evidence has shown an association between organochlorine compounds (OCC) exposure and human health hazards. Concerning this, OCC detection in human adipose samples has to be considered a public health priority. This study evaluated the efficacy of various solid-phase extraction (SPE) and cleanup methods for OCC determination in human adipose tissue. Octadecylsilyl endcapped (C18-E), benzenesulfonic acid modified silica cation exchanger (SA), poly (styrene-divinylbenzene (EN) and EN/RP18 SPE sorbents were evaluated. The relative sample cleanup provided by these SPE columns was evaluated using gas chromatography with electron capture detection (GC–ECD). The C18-E columns with strong homogenization were found to provide the most effective cleanup, removing the greatest amount of interfering substance, and simultaneously ensuring good analyte recoveries higher than 70%. Recoveries>70% with standard deviations (SD)<15% were obtained for all compounds under the selected conditions. Method detection limits were in the 0.003–0.009 mg/kg range. The positive samples were confirmed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). The highest percentage found of the OCC in real samples corresponded to HCB, o,p′-DDT and methoxychlor, which were detected in 80 and 95% of samples analyzed respectively. Copyright © 2012 John Wiley & Sons, Ltd.

Interlaboratory validation of an environmental monitoring method for trace analysis of endocrine disrupting compounds

Environmental pollution continues to be an emerging study field, as there are thousands of anthropogenic compounds mixed in the environment whose possible mechanisms of toxicity and physiological outcomes are of great concern. Developing methods to access and prioritize the screening of these compounds at trace levels in order to support regulatory efforts is, therefore, very important. A methodology based on solid phase extraction followed by derivatization and gas chromatography-mass spectrometry analysis was developed for the assessment of four endocrine disrupting compounds (EDCs) in water matrices: bisphenol A, estrone, 17b-estradiol and 17a-ethinylestradiol. The study was performed, simultaneously, by two different laboratories in order to evaluate the robustness of the method and to increase the quality control over its application in routine analysis. Validation was done according to the International Conference on Harmonisation recommendations and other international guidelines with specifications for the GC-MS methodology. Matrix-induced chromatographic response enhancement was avoided by using matrix-standard calibration solutions and heteroscedasticity has been overtaken by a weighted least squares linear regression model application. Consistent evaluation of key analytical parameters such as extraction efficiency, sensitivity, specificity, linearity, limits of detection and quantification, precision, accuracy and robustness was done in accordance with standards established for acceptance. Finally, the application of the optimized method in the assessment of the selected analytes in environmental samples suggested that it is an expedite methodology for routine analysis of EDC residues in water matrices.

Simultaneous distillation–extraction of high-value volatile compounds from Cistus ladanifer L.

The present paper describes a procedure to isolate volatiles from rock-rose (Cistus ladanifer L.) using simultaneous distillation–extraction (SDE). High-value volatile compounds (HVVC) were selected and the influence of the extraction conditions investigated. The effect of the solvent nature and extraction time on SDE efficiency was studied. The best performance was achieved with pentane in 1 h operation. The extraction efficiencies ranged from 65% to 85% and the repeatability varied between 4% and 6% (as a CV%). The C. ladanifer SDE extracts were analysed by headspace solid phase microextraction (HS-SPME) followed by gas chromatography with flame ionization detection (GC-FID). The HS-SPME sampling conditions such as fiber coating, temperature, ionic strength and exposure time were optimized. The best results were achieved with an 85 µm polyacrylate fiber for a 60 min headspace extraction at 40ºC with 20% (w/v) of NaCl. For optimized conditions the recovery was in average higher than 90% for all compounds and the intermediate precision ranged from 4 to 9% (as CV %). The volatiles α-pinene (22.2 mg g−1 of extract), 2,2,6-trimethylcyclohexanone (6.1 mg g−1 of extract), borneol (3.0 mg g−1 of extract) and bornyl acetate (3.9 mg g−1 of extract) were identified in the SDE extracts obtained from the fresh plant material.

Exploring bioactive properties of marine cyanobacteria Isolated from the Portuguese coast: high potential as a source of anticancer compounds

The oceans remain a major source of natural compounds with potential in pharmacology. In particular, during the last few decades, marine cyanobacteria have been in focus as producers of interesting bioactive compounds, especially for the treatment of cancer. In this study, the anticancer potential of extracts from twenty eight marine cyanobacteria strains, belonging to the underexplored picoplanktonic genera, Cyanobium, Synechocystis and Synechococcus, and the filamentous genera, Nodosilinea, Leptolyngbya, Pseudanabaena and Romeria, were assessed in eight human tumor cell lines. First, a crude extract was obtained by dichloromethane:methanol extraction, and from it, three fractions were separated in a Si column chromatography. The crude extract and fractions were tested in eight human cancer cell lines for cell viability/toxicity, accessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactic dehydrogenase release (LDH) assays. Eight point nine percent of the strains revealed strong cytotoxicity; 17.8% showed moderate cytotoxicity, and 14.3% assays showed low toxicity. The results obtained revealed that the studied genera of marine cyanobacteria are a promising source of novel compounds with potential anticancer activity and highlight the interest in also exploring the smaller filamentous and picoplanktonic genera of cyanobacteria.

Brewer's spent grain from different types of malt: Evaluation of the antioxidant activity and identification of the major phenolic compounds

The antioxidant activity and phenolic composition of brewer's spent grain (BSG) extracts obtained by microwave-assisted extraction from twomalt types (light and darkmalts) were investigated. The total phenolic content (TPC) and antioxidant activity among the light BSG extracts (pilsen, melano, melano 80 and carared)were significantly different (p b 0.05) compared to dark extracts (chocolate and black types), with the pilsen BSG showing higher TPC (20 ± 1 mgGAE/g dry BSG). In addition, the antioxidant activity assessed by 2,2-diphenyl- 1-picrylhydrazyl, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and deoxyribose assays decreased as a result of increasing kilning temperatures in the following order: pilsen N melano N melano 80 N carared N chocolate N black. HPLC-DAD/ESI-MS/MS analysis indicated the presence of phenolic acids, such as ferulic, p-coumaric and syringic acids, as well as several isomeric ferulate dehydrodimers and one dehydrotrimer. Chocolate and black extracts, obtained frommalts submitted to the highest kilning temperatures, showed the lowest levels of ferulic and p-coumaric acids. These results suggested that BSG extracts from pilsen malt might be used as an inexpensive and good natural source of antioxidants with potential interest for the food, pharmaceutical and/or cosmetic industries after purification.

Project, modelling and simulation of air stripping columns for the treatment of groundwater contaminated with volatile organic compounds

Volatile organic compounds are a common source of groundwater contamination that can be easily removed by air stripping in columns with random packing and using a counter-current flow between the phases. This work proposes a new methodology for the column design for any particular type of packing and contaminant avoiding the necessity of a pre-defined diameter used in the classical approach. It also renders unnecessary the employment of the graphical Eckert generalized correlation for pressure drop estimates. The hydraulic features are previously chosen as a project criterion and only afterwards the mass transfer phenomena are incorporated, in opposition to conventional approach. The design procedure was translated into a convenient algorithm using C++ as programming language. A column was built in order to test the models used either in the design or in the simulation of the column performance. The experiments were fulfilled using a solution of chloroform in distilled water. Another model was built to simulate the operational performance of the column, both in steady state and in transient conditions. It consists in a system of two partial non linear differential equations (distributed parameters). Nevertheless, when flows are steady, the system became linear, although there is not an evident solution in analytical terms. In steady state the resulting system of ODE can be solved, allowing for the calculation of the concentration profile in both phases inside the column. In transient state the system of PDE was numerically solved by finite differences, after a previous linearization.

Practical software improve design and performance of a randomly packed air stripping column for removing volatile organic compounds from water

STRIPPING is a software application developed for the automatic design of a randomly packing column where the transfer of volatile organic compounds (VOCs) from water to air can be performed and to simulate it’s behaviour in a steady-state. This software completely purges any need of experimental work for the selection of diameter of the column, and allows a choice, a priori, of the most convenient hydraulic regime for this type of operation. It also allows the operator to choose the model used for the calculation of some parameters, namely between the Eckert/Robbins model and the Billet model for estimating the pressure drop of the gaseous phase, and between the Billet and Onda/Djebbar’s models for the mass transfer. Illustrations of the graphical interface offered are presented.

A New Approach in the Design of Air Stripping Columns for the Treatment of Groundwater Contaminated With Volatile Organic Compounds

Volatile organic compounds are a common source of groundwater contamination that can be easily removed by air stripping in columns with random packing and using a counter-current flow between the phases. This work proposes a new methodology for column design for any type of packing and contaminant which avoids the necessity of an arbitrary chosen diameter. It also avoids the employment of the usual graphical Eckert correlations for pressure drop. The hydraulic features are previously chosen as a project criterion. The design procedure was translated into a convenient algorithm in C++ language. A column was built in order to test the design, the theoretical steady-state and dynamic behaviour. The experiments were conducted using a solution of chloroform in distilled water. The results allowed for a correction in the theoretical global mass transfer coefficient previously estimated by the Onda correlations, which depend on several parameters that are not easy to control in experiments. For best describe the column behaviour in stationary and dynamic conditions, an original mathematical model was developed. It consists in a system of two partial non linear differential equations (distributed parameters). Nevertheless, when flows are steady, the system became linear, although there is not an evident solution in analytical terms. In steady state the resulting ODE can be solved by analytical methods, and in dynamic state the discretization of the PDE by finite differences allows for the overcoming of this difficulty. To estimate the contaminant concentrations in both phases in the column, a numerical algorithm was used. The high number of resulting algebraic equations and the impossibility of generating a recursive procedure did not allow the construction of a generalized programme. But an iterative procedure developed in an electronic worksheet allowed for the simulation. The solution is stable only for similar discretizations values. If different values for time/space discretization parameters are used, the solution easily becomes unstable. The system dynamic behaviour was simulated for the common liquid phase perturbations: step, impulse, rectangular pulse and sinusoidal. The final results do not configure strange or non-predictable behaviours.

Organic versus conventional tomatoes: Influence on physicochemical parameters, bioactive compounds and sensorial attributes

The effect of organic and conventional agricultural systems on the physicochemical parameters, bioactive compounds content, and sensorial attributes of tomatoes (‘‘Redondo’’ cultivar) was studied. The influence on phytochemicals distribution among peel, pulp and seeds was also accessed. Organic tomatoes were richer in lycopene (+20%), vitamin C (+30%), total phenolics (+24%) and flavonoids (+21%) and had higher (+6%) in vitro antioxidant activity. In the conventional fruits, lycopene was mainly concentrated in the pulp, whereas in the organic ones, the peel and seeds contained high levels of bioactive compounds. Only the phenolic compounds had a similar distribution among the different fractions of both types of tomatoes. Furthermore, a sensorial analysis indicated that organic farming improved the gustative properties of this tomato cultivar.

Novel LTCC-potentiometric microfluidic device for biparametric analysis of organic compounds carrying plastic antibodies as ionophores: Application to sulfamethoxazole and trimethoprim

Monitoring organic environmental contaminants is of crucial importance to ensure public health. This requires simple, portable and robust devices to carry out on-site analysis. For this purpose, a low-temperature co-fired ceramics (LTCC) microfluidic potentiometric device (LTCC/μPOT) was developed for the first time for an organic compound: sulfamethoxazole (SMX). Sensory materials relied on newly designed plastic antibodies. Sol–gel, self-assembling monolayer and molecular-imprinting techniques were merged for this purpose. Silica beads were amine-modified and linked to SMX via glutaraldehyde modification. Condensation polymerization was conducted around SMX to fill the vacant spaces. SMX was removed after, leaving behind imprinted sites of complementary shape. The obtained particles were used as ionophores in plasticized PVC membranes. The most suitable membrane composition was selected in steady-state assays. Its suitability to flow analysis was verified in flow-injection studies with regular tubular electrodes. The LTCC/μPOT device integrated a bidimensional mixer, an embedded reference electrode based on Ag/AgCl and an Ag-based contact screen-printed under a micromachined cavity of 600 μm depth. The sensing membranes were deposited over this contact and acted as indicating electrodes. Under optimum conditions, the SMX sensor displayed slopes of about −58.7 mV/decade in a range from 12.7 to 250 μg/mL, providing a detection limit of 3.85 μg/mL and a sampling throughput of 36 samples/h with a reagent consumption of 3.3 mL per sample. The system was adjusted later to multiple analyte detection by including a second potentiometric cell on the LTCC/μPOT device. No additional reference electrode was required. This concept was applied to Trimethoprim (TMP), always administered concomitantly with sulphonamide drugs, and tested in fish-farming waters. The biparametric microanalyzer displayed Nernstian behaviour, with average slopes −54.7 (SMX) and +57.8 (TMP) mV/decade. To demonstrate the microanalyzer capabilities for real applications, it was successfully applied to single and simultaneous determination of SMX and TMP in aquaculture waters.