Comparison of diode array and electrochemical detection in the C30 reverse phase HPLC analysis of algae carotenoids

Qualitative and quantitative determination of carotenoids pigments can provide valuable information about the organisms in which this important class of compounds is found. In the HPLC analysis of pigments, diode array (DAD), electrochemical (ED) and other kinds of detector may be used. The aim of this work is to develop an HPLC method using a C30 column to identify and quantify sixteen different pigments from algae. A further aim is to compare precision and accuracy obtained by DAD and ED. ED is normally more sensible than DAD. On the other hand, the highest precision and accuracy was obtained with DAD. In conclusion, the method was efficient for quantitative and qualitative analyses of pigments from cyanobacteria and different microalgae classes. Their pigment patterns for several organisms are also discussed.A determinação qualitativa e quantitativa de carotenóides pode fornecer diferentes e importantes informações sobre os organismos que os contêm. Na análise de pigmentos por HPLC diversos detectores podem ser utilizados, como diode array (DAD) e eletroquímico (ED). O presente trabalho tem como objetivo desenvolver um método por HPLC utilizando uma coluna C30 para a identificação e quantificação de dezesseis pigmentos em diferentes classes de algas, além de comparar as respostas obtidas nos detectores DAD e ED por meio da análise dos resultados de precisão e exatidão. Apesar do ED ser geralmente um detector mais sensível que o DAD, os resultados de precisão e exatidão foram mais satisfatórios para o DAD. O método desenvolvido foi eficiente para a análise quantitativa dos pigmentos de cianobactérias e diferentes classes de algas, sendo que o padrão cromatográfico encontrado em cada classe foi discutido.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Milênio-Redoxom

Astaxanthin Restrains Nitrative-Oxidative Peroxidation in Mitochondrial-Mimetic Liposomes: A Pre-Apoptosis Model

Astaxanthin (ASTA) is a ketocarotenoid found in many marine organisms and that affords many benefits to human health. ASTA is particularly effective against radical-mediated lipid peroxidation, and recent findings hypothesize a "mitochondrial-targeted" action of ASTA in cells. Therefore, we examined the protective effects of ASTA against lipid peroxidation in zwitterionic phosphatidylcholine liposomes (PCLs) and anionic phosphatidylcholine: phosphatidylglycerol liposomes (PCPGLs), at different pHs (6.2 to 8.0), which were challenged by oxidizing/nitrating conditions that mimic the regular and preapoptotic redox environment of active mitochondria. Pre-apoptotic conditions were created by oxidized/nitr(osyl) ated cytochrome c and resulted in the highest levels of lipoperoxidation in both PCL and PCPGLs (pH 7.4). ASTA was less protective at acidic conditions, especially in anionic PCPGLs. Our data demonstrated the ability of ASTA to hamper oxidative and nitrative events that lead to cytochrome c-peroxidase apoptosis and lipid peroxidation, although its efficiency changes with pH and lipid composition of membranes.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESPBPE fellowship)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (Bolsa Produtividade em Pesquisa, Nivel 2, CNPq, Brazil)Programa Iberoamericano de Ciencia y Tecnologia para el Desarollo (CYTEDRed iberoamericana para el estudio de nuevos carotenoides bioactivos como ingredientes de alimentos, Spain)Univ Sao Paulo IQUSP, Inst Quim, Dept Bioquim, BR-05508000 Sao Paulo, SP, BrazilUniv Cruzeiro Sul, ICAFE, BR-01506000 Sao Paulo, SP, BrazilSuperintendencia Policia Tecn Cient, BR-05507060 Sao Paulo, SP, BrazilLychnoflora Pesquisa & Dev Prod Nat LTDA, BR-14030090 Ribeirao Preto, SP, BrazilGrp Fleury, BR-04344070 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Ciencias Exatas & Terra, UNIFESP, BR-09972270 Diadema, SP, BrazilUniv Sao Paulo IQUSP, Inst Quim, Dept Quim Fundamental, BR-05508000 Sao Paulo, SP, BrazilCSIC, IATA, Dept Ciencia Alimentos, Calle Catedrat Agustin Escardino 7, Paterna 46980, SpainUniv Fed Sao Paulo, Dept Ciencias Exatas & Terra, UNIFESP, BR-09972270 Diadema, SP, BrazilFAPESP: 017/06032-2CNPq: 304663/2015-8RIENCBI: 112RT0445Web of Scienc

A Fragmentation study of di-acidic mycosporine-like amino acids in electrospray and nanospray mass spectrometry

Two mycosporine (MAAs), containing an extra acid function, were analyzed by nanospray and electrospray ionization tandem mass spectrometry. In contrast to the previous studies it is demonstrated that no significant characteristic methyl radical loss occurred in positive mode. The fragmentation pathway in negative mode was also proposed in this work, along with theoretical calculations to characterize the site of protonation.No presente estudo, duas micosporinas (MAAs) contendo um segundo ácido carboxílico foram submetidas à fragmentação em eletrospray e nanospray em diferentes equipamentos. Em contraste com resultados anteriores, a eliminação de radical metila no modo positivo de análise foi um processo minoritário de fragmentação. Neste trabalho apresentamos também a via de fragmentação destas substâncias em modo negativo e cálculos teóricos para caracterizar os sítios de protonação.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Fragmentation study of di-acidic mycosporine-like amino acids in electrospray and nanospray mass spectrometry

No presente estudo, duas micosporinas (MAAs) contendo um segundo ácido carboxílico foram submetidas à fragmentação em eletrospray e nanospray em diferentes equipamentos. Em contraste com resultados anteriores, a eliminação de radical metila no modo positivo de análise foi um processo minoritário de fragmentação. Neste trabalho apresentamos também a via de fragmentação destas substâncias em modo negativo e cálculos teóricos para caracterizar os sítios de protonação. Two mycosporine (MAAs), containing an extra acid function, were analyzed by nanospray and electrospray ionization tandem mass spectrometry. In contrast to the previous studies it is demonstrated that no significant characteristic methyl radical loss occurred in positive mode. The fragmentation pathway in negative mode was also proposed in this work, along with theoretical calculations to characterize the site of protonation. Keywords: mycosporine, nanoESI-MS/MS, ESI-MS/MS, natural products, algae Introduction Mycosporine-like amino acids (MAAs) are a group of chemically related, water soluble compounds responsible for UV photoprotection in a diverse range of organisms including invertebrates, fish, bacteria, cyanobacteria, micro-and macroalgae. 1-3 These compounds are chemically characterised by the presence of either a cyclohexenone or cyclohexenimine chromophore conjugated with a substituent nitrogen of an amino acid, amino alcohol or amino group 1 MAAs normally show a strong UV absorption between 310 and 360 nm with high molar extinction coefficients. These characteristics indicate a possible photoprotective role that has been demonstrated in a number of studies 11-13 17 In the last few years, nanospray ionisation (nanoESI) is beginning to increase in importance, especially with the development of automated systems using 'chips' (arrays of uniform nanospray needles that are used only once to avoid contamination). 21 NanoESI offers the possibility of improved sensitivity and lower sample consumption over conventional ESI for the analysis of natural products. 22 This is especially important for the study of extracts from biological and medicinal sources when often only a very small amount of material is available. In 'chip based' nanoESI, the analyte solution is sprayed from a conducting pipette tip pressed against the rear of a chip using a small gas pressure and much lower voltages to create the spray. Recently, analysis of some natural antioxidants (retinoids Experimental Chemicals All solvents used were HPLC grade (Tedia, J. Baker and Fisher). Water was purified using a Milli-Q system (Millipore, Bedford, MA, USA). Trifluoroacetic acid (99.9%) was purchased from Aldrich. Galena Química e Farmacêutica Ltda/Brazil kindly supplied the standards of shinorine and porphyra-334 (product Helioguard ® 365-Porphyra umbilicalis extracts) Instrumentation Nanospray ionisation analyses were performed on two quadrupole-time of flight hybrid instruments: (a) an UltrOTOF-Q (Bruker Daltonics, Billerica, MA) using Tip™ Emittek (glass tip capillaries working with 500 V) or (b) a QStar-XL (Applied Biosystems, Warrington, UK) using a Nanomate HD automatic 'chip based' nanospray system (Advion Biosciences, Norwich, UK). The Nanomate was set for 5 µL of solution to be aspirated and sprayed through a Nanomate 400 chip at 1.45 kV, with a nitrogen back pressure of 0.4 psi. On both instruments, the ion source gas and curtain gas were nitrogen. Electrospray ionisation analyses were performed on five instruments: (a) an Apex 4 7.0 Tesla Fouriertransform ion-cyclotron resonance mass spectrometer (Bruker Daltonics, Billerica, MA, USA). Samples were directly infused into the Apollo electrospray source from a syringe pump at 100 µL h -1 . Analyses were performed at a capillary voltage, of 4600V and capillary exit potential of 200 V (except were indicated otherwise). The N 2 drying gas temperature was 200 °C. A mixture of PEG grades was used as an external calibrant for accurate-mass ESI analysis; (b) on a quadrupole-time of flight instrument (UltrOTOF-Q, Bruker Daltonics, Billerica, MA). The analyses were performed in positive ion ESI mode at a capillary voltage of 3400 V and N 2 drying gas temperature of 180 °C. NaTFA 10 mmol L -1 was used as a standard for internal and external calibration; (c) on a QStar-XL quadrupole-time-of-flight instrument (Applied Biosystems, Warrington, UK); (d) on a Quattro-LC triple quadrupole mass spectrometer (Micromass, Manchester, UK); (e) on an Esquire HCT ion trap instrument (Bruker Daltonics, Billerica, MA, USA) using a syringe pump (Cole-Parmer, Vernon Hills, IL, USA). Ion trap analyses were performed using nitrogen as the nebulising and drying gas and helium as the bath gas (4×10 -6 mbar). Theoretical calculations All calculations were performed in Gaussian 03 27 suite of programs using the B3LYP/6-31+G(d,p) model. 28,29 The geometries of neutral and protonated species were optimised and the potential energy surface minima were indicated by analysis of vibrational frequencies. The gasphase basicity and proton affinity were calculated via a protonation reaction, using Gibbs energies and enthalpies, respectively. 30 Cardozo et al. 1627 Vol. 20, No. 9, 2009 Results and Discussion Mycosporine-like amino acids with one acidic function were previously analysed by positive mode ESI sequential mass spectrometry (at high-resolution and accurate-mass). The loss of a methyl radical by the homolytic cleavage of the O-C bond was observed to be the preferred fragmentation pathway. As expected, analysis in negative ion mode Conclusions These results indicate that the presence of a second carboxylic acid function significantly reduces the intensity of the observed product ions from the radical methyl cleavage in positive mode MS/MS. As expected, in the negative ion mode, the radical fragmentation pathway does not occur. Taken together, these results confirm the importance of careful selection of the product ions used for analytical protocols for the analysis of crude extracts containing MAAs where the presence of the second acid function may change the fragmentation behavior and the classical analysis of loss of methyl radical may lead to the wrong conclusions during screening for MAAs

Palythine-threonine, a major novel mycosporine-like amino acid (MAA) isolated from the hermatypic coral Pocillopora capitata

Using a high-resolution reverse-phase liquid chromatography method we found that the tissues of the hermatypic coral Pocillopora capitato (collected in Santiago Bay, Mexico) contain a high diversity of primary and secondary mycosporine-like amino acids (MAAs) typical of some reef-building coral species: mycosporine-glycine, shinorine, porphyra-334, mycosporine-methylamine-serine, mycosporine-methylamine-threonine, palythine-serine, palythine and one additional novel predominant MAA, with an absorbance maximum of 320 nm. Here we document the isolation and characterization of this novel MAA from the coral A capitata. Using low multi-stage mass analyses of deuterated and non deuterated compounds, high-resolution mass analyses (Time of Flight, TOF) and other techniques, this novel compound was characterized as palythine-threonine. Palythine-threonine was also present in high concentrations in the corals Pocillopora eydouxi and Stylophora pistillata indicating a wider distribution of this MAA among reef-building corals. From structural considerations we suggest that palythine-threonine is formed by decarboxylation of porphyra-334 followed by demethylation of mycosporine-methylamine-threonine. (C) 2008 Elsevier B.V. All rights reserved.CNPq-Instituto do Milenio and Fapesp from BrazilCNPq-Instituto do MilenioFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CONICET from ArgentinaCONICET from Argentina[PIP 5009]INIDEP[1535]INIDE

Analyses of photoprotective compounds in red algae from the Brazilian coast

Qualitative and quantitative studies of mycosporine-like amino acids (MAAs) in three species of the genus Gracilaria Greville (G. birdiae, G. domingensis and G. tenuistipitata) were performed. A simple and efficient extraction procedure based on ethanol was described. HPLC, UV and mass spectrometry experiments revealed different profiles between extracts obtained from one species cultivated in the laboratory (G. tenuistipitata) and two species collected in their natural environment (G. birdiae and G. domingensis). The levels detected in the latter two species were approximately 150 times higher than in the species cultivated in vitro. This study revealed that G. birdiae and G. domingensis present a potential source for economical exploration of MAAs

Trust and reputation for critical infrastructure protection

Atualmente a sociedade contemporânea tem ao seu dispor um sem numero de serviços que suportam toda a economia globalizada em que vivemos bem como o nosso modo de vida. Serviços como distribuição de energia, água, gás, redes de transportes, telecomunicações, a Internet, entre outros, são atualmente parte integrante da vida dos cidadãos e das empresas. Estes serviços estão de tal forma presentes nas nossas vidas que a sua relevância e o grau de dependência aos serviços, apenas é sentido aquando da sua indisponibilidade. Este tipo de serviço dos quais depende o nosso modo de vida, são fornecidos por infraestruturas críticas, assim referidas pois a sua falha ou quebra da qualidade do serviço prestado pode ter um grande impacto na sociedade ou economia de um País. Para além dos fenómenos da natureza e dos riscos inerentes à sua própria exploração, os riscos que estas infraestruturas correm têm vindo a aumentar ao atrair cada vez mais o interesse de grupos de hackers e terroristas, principalmente pela forte visibilidade e consequências que mesmo um pequeno ataque pode acarretar. De entre os problemas inerentes ao funcionamento das infraestruturas críticas destaca-se o fato da existência de dependências ou interdependências entre infraestruturas. Veja-se o exemplo do serviço de telecomunicações que está por natureza dependente do fornecimento de energia elétrica ou dos serviços bancários que estão dependentes de ambos. Mas não está atualmente o fornecimento de energia dependente dos serviços de telecomunicações e dos seus sistemas de informação? Destes exemplos torna-se visível que, para além da (inter)dependência que possa existir, é necessário analisar também os efeitos em cascata que podem surgir após a falha de uma infraestrutura. Com o objetivo de promover a segurança em infraestruturas críticas, vários governos, em conjunto com a comunidade científica, promovem esforços de investigação nesta área. Em particular, nas áreas da distribuição de energia e das telecomunicações. Ao nível da União Europeia, existe grande determinação para promover projetos nesta área, em particular, projetos que promovem a troca de informação entre infraestruturas, na forma de alertas de risco, prevenindo os Operadores das infraestruturas relativamente a um aumento de risco de perda ou quebra de qualidade do serviço fornecido. Esta troca permite que as infraestruturas possam aplicar atempadamente os seus planos de contingência ou recuperação, minimizando eventuais quebras de serviço e consequentemente reduzindo o indesejado efeito de falha em cascata. A motivação para o trabalho apresentado nesta tese, surgiu da identificação dos principais aspectos em aberto relativos à troca e gestão de alertas de risco entre infraestruturas críticas. Muitas das abordagens existentes relativas à segurança em infraestruturas críticas focam-se na obtenção de níveis de risco através do uso de modelos mais ou menos complexos das infraestruturas. Apesar de estes modelos permitirem uma base sólida para a monitorização do risco, não apresentam mecanismos para a sua troca, gestão e avaliação de qualidade. Este trabalho aborda o problema relacionado com a confiança, reputação e gestão de alertas de risco no seio das infraestruturas críticas. Nesse sentido é proposta a introdução de mecanismos que permitam gerir e aferir em cada instante, o grau de confiança atribuído a cada um dos alertas de risco recebidos ou calculados internamente, permitindo melhorar a sua precisão e consequentemente melhorar também a resiliência da infraestrutura critica quando confrontada com alertas de riscos imprecisos ou inconsistentes. Na tese é abordado o problema da segurança em infraestruturas críticas interdependentes e identificados os principais problemas inerentes à troca de informação de risco, em particular, a forma de efetuar a partilha de informação de uma forma segura, a gestão dessa mesma partilha e a avaliação da fiabilidade da informação envolvida na partilha. Propõe-se nesta tese, a aplicação de mecanismos de gestão baseados no paradigma de gestão por politicas para a gestão da partilha de alertas de risco entre infraestruturas críticas. Com o objetivo de melhorar a gestão da partilha e posterior interpretação dos alertas de risco, é proposta a introdução da análise de confiança e reputação na avaliação da fiabilidade da informação envolvida na partilha e na avaliação do comportamento das entidades envolvidas. As propostas apresentadas nesta tese são discutidas e aplicadas no âmbito do projeto Europeu MICIE (Tool for systemic risk analysis and secure mediation of data exchanged across linked CI information infrastructures), em particular, no que se refere à solução proposta para a gestão da partilha de alertas de risco, que em conjunto com os indicadores de confiança e reputação propostos, permitem melhorar a proteção de cada infraestrutura relativamente ao uso de informação menos confiável ou inconsistente. Apresenta-se também a adaptação dos conceitos propostos ao CI Security Model, um modelo de análise de risco em tempo real, no qual as falhas identificadas são atenuadas com a introdução da análise de confiança e reputação proposta nesta tese. Os resultados da avaliação das propostas apresentadas são discutidos com base em cenários de simulação bem como através de dados reais de uma infraestrutura crítica. Os resultados obtidos indicam que as propostas apresentadas satisfazem os objectivos definidos, nomeadamente, ao contribuir para o aumento da confiança que uma infraestrutura crítica tem relativamente à informação recebida em tempo real acerca dos serviços dos quais depende, ao permitir uma melhor gestão dessa mesma informação e também ao contribuir para o aumento da fiabilidade dos resultados provenientes dos modelos de risco em uso na infraestrutura.Currently, our society has at its disposal an uncountable number of services able to support the global economy and also our current way of life. Services such as power distribution, water, gas, transport networks, telecommunications, the Internet, among others, are now an integral part of the citizens' lives and businesses. These services play such a big role in our lives that their importance is only appreciated when they are unavailable. These types of services, that our lives so heavily depend on, are provided by Critical Infrastructures. They are referred to as ``Critical" due to the fact that in case of failure or breakdown in providing quality of service, the impact on society and the economy of a country can be enormous. Beyond the phenomena of nature and risks inherent to the infrastructure operation, the risks faced by these infrastructures have continuously increasing, by attracting interest from groups of hackers and terrorist groups. Primarily due to the strong visibility and consequences that may result even from a small successful attack. Among the problems inherent to the operation of Critical Infrastructures, it is possible to emphasise the existence of dependencies and interdependencies among infrastructures. For example, a telecommunications service is inherently dependent on the electricity supply or, for instance, banking services are dependent on both telecommunications and energy supply services. However, is it not the service that provides power supply actually dependent on telecommunications services and also on information systems? Based on these examples it becomes apparent that in addition to the (inter)dependence that may exist, it is also necessary to examine the cascading effects that may arise after the failure of a Critical Infrastructure. Critical Infrastructures security has been the subject of discussion by numerous governments with the support of the academia by promoting research efforts in these areas, in particular in areas such as power distribution and telecommunications. Furthermore, within the European Union, there is determination to promote projects in these areas, in particular the promotion of projects that foster the exchange of information, in the form of warnings, among infrastructures. These warnings allow the Critical Infrastructure to be informed and aware of the increasing risk of loss or reduction in quality of the service received. This exchange allows the infrastructure to timely implement their contingency and recovery plans to minimise any service breaks and consequently minimise the unwanted effect of a cascading failure. The motivation for the work presented in this thesis arose from the identification of the main open issues relating to the exchange and management of risk warnings among Critical Infrastructures. Many of the existing approaches to security in Critical Infrastructures are focused on obtaining risk levels through the use of models based on the infrastructure. Although these models allow a solid foundation for risk monitoring, they do not have mechanisms for exchange, management and assessment of its quality. This work addresses the problem related to trust, reputation and risk alerts management within Critical Infrastructures. Accordingly, it is proposed to introduce mechanisms to manage and measure at each instant, the degree of confidence assigned to each of the alerts received or computed internally. Allowing improvement of their accuracy and consequently improving the resilience of Critical Infrastructures when faced with inaccurate or inconsistent risk alerts. This thesis addresses the problem of interdependent Critical Infrastructure security and identifies the main problems related to risk information sharing. In particular, how to allow information sharing in a secure manner, the management of that sharing and how to assess the reliability of such information. This thesis proposes the application of Policy Based Management mechanisms for the management of the risk alert information shared among Critical Infrastructures. In order to improve the information sharing management and the further interpretation of the risk alerts, it is proposed to evaluate Trust and Reputation in order to assess the shared information and also to consider the behaviour of the entities involved. The proposals presented in this thesis are discussed and applied in the context of the European Project MICIE ({Tool for systemic risk analysis and secure mediation of data exchanged across linked CI information infrastructures). In particular with regard to the proposed solution for the management of shared risk alerts, which uses the Policy Based Management paradigm. By incorporating the proposed Trust and Reputation indicators it allows to improve the Critical Infrastructure protection considering the use of untrustworthy or inconsistent information. It is also proposed the adaptation of the presented concepts to the CI Security Model, a model for real time risk analysis evaluation, in which the identified shortcomings are addressed with the integration of the Trust and Reputation approach proposed in this thesis. The results of the proposals evaluation are discussed based on simulation scenarios as well as through real data of a Critical Infrastructure. The achieved results indicate that the proposed mechanisms meet the objectives such as, by contributing to the increase in confidence that a Critical Infrastructure has on the information received about the services on which it depends. To allow improvement in management of such information as well as contribution to increased reliability of results obtained from the risk models applied to the infrastructure.Fundação para a Ciência e a Tecnologia (FCT