Res Antiquitatis. Journal of Ancient History

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Determination of antipsychotic drugs in oral fluid samples using dried saliva spots

The present work describes the optimization and validation of an analytical method for the determination of six antipsychotic drugs (chlorpromazine, levomepromazine, cyamemazine, clozapine, haloperidol and quetiapine) in oral fluid samples after solvent extraction from dried saliva spots, by gas chromatography coupled to tandem mass spectrometry. The method was fully validated, and the included parameters were selectivity, linearity, limits of quantification, precision and accuracy, stability and recovery. The method was linear for all compounds from 10-400 ng/mL, except for haloperidol (5-100 ng/mL), presenting coefficients of determination higher than 0.99. Inter- and intra-day precision and accuracy were in conformity with the criteria usually seen in bioanalytical method validation, i.e., coefficients of variation were lower than 15% and an accuracy of 15% or better for all studied drugs. The recoveries obtained with this miniaturized technique ranged from 63 to 97%. The herein described method is the first to be reported using the dried saliva spots approach for the analysis of these antipsychotic drugs, proving great sensitivity apart from its simple and fast procedure. The method was considered a good alternative to the conventional techniques to be applied in clinical and toxicological analyses, even more taking into account the extremely low sample volume used (50 µL)A esquizofrenia é considerada uma das doenças mentais que mais incapacidade causa em todo o mundo, afetando 1% da população. São múltiplas as causas que podem dar origem a esta doença, tais como fatores bioquímicos, hereditários, entre outros. Atualmente não se dispõe de uma cura mas a minimização dos seus sintomas é conseguida através da administração de medicamentos e/ou terapias psicossociais. Nos últimos anos, o tratamento desta doença mental teve um grande impacto na área da investigação, visto que o número de suicídios em relação a pacientes esquizofrénicos foi aumentando ligeiramente. O tratamento da esquizofrenia é realizado através de medicação antipsicótica que tem como objetivo principal atenuar os sintomas psicóticos. Consoante o diagnóstico do paciente, é importante que exista uma vigilância na administração de antipsicóticos de forma a evitar overdoses ou erros na medicação. Segundo o relatório da INFARMED, o consumo de antipsicóticos em Portugal entre 2000 e 2012 aumentou, tendo como principais causas a utilização prolongada destes fármacos e a facilidade com que se acede a estes. Estes tópicos levantam um importante problema de saúde pública, contribuindo para o desenvolvimento de investigações na área toxicológica e clínica. Em relação ao desenvolvimento de técnicas analíticas para a deteção e quantificação dos antipsicóticos, a amostra de fluido oral tem vindo a ter um grande impacto devido à sua facilidade de recolha e por ser considerada uma amostra não invasiva. Ultimamente, como técnica de extração de amostra, os dried saliva spots (DSS) têm sido utilizados na deteção de diversos tipos de fármacos, visto ser um processo que requer um baixo volume de saliva e o seu processo ser bastante simples e rápido. Tendo por base esta premissa o presente trabalho descreve o desenvolvimento, otimização e validação de um método analítico para a determinação de seis antipsicóticos (clorpromazina, levomepromazina, ciamemazina, clozapina, haloperidol e quetiapina) em saliva com recurso a DSS e a cromatografia gasosa acoplada à espectrometria de massas em tandem (GC-MS/MS). Os fármacos estudados foram selecionados com base na sua prescrição e associação a efeitos tóxicos. Os padrões internos utilizados foram a clorpromazina-d3 e a promazina, visto que apresentam características químicas semelhantes às dos compostos em estudo. O método proposto foi totalmente validado quanto a seletividade, linearidade, limites de quantificação, precisão e exatidão, estabilidade e recuperação conforme os critérios internacionais sugerem. A otimização do processo de extração foi realizada de acordo com os seguintes parâmetros: escolha do solvente mais adequado (metanol acidificado), volume do solvente (2 mL), bem como o tempo de extração (5 minutos) e o tempo de secagem das amostras de fluido oral aplicadas nos DSS (1 hora). O método foi linear para todos os compostos numa gama de concentrações de 10-400 ng/mL, exceto para o haloperidol (5-100 ng/mL), apresentando coeficientes de determinação superiores a 0.99. A precisão e exatidão inter- e intra-dia foram realizadas de acordo com a validação bioanalítica do método, isto é, os coeficientes de variação apresentam-se abaixo dos 15% e uma exatidão de ± 15% ou inferior, para todos os fármacos em estudo. As recuperações obtidas com esta técnica miniaturizada variaram entre 63% a 97%. De forma a verificar a fiabilidade do método, este foi aplicado a amostras de fluido oral procedentes de doentes em tratamento com estes fármacos. Salienta-se que o método que aqui se descreve é o primeiro trabalho que utiliza DSS como técnica de extração para a análise de antipsicóticos. Os resultados apresentados permitem afirmar que a técnica proposta apresenta uma excelente sensibilidade (5-10 ng/mL) para além de ser um procedimento que a nível prático mostra uma maior simplicidade e rapidez comparativamente aos outros métodos de extração. Por outro lado, o volume de amostra constitui uma grande vantagem, visto que se utiliza apenas 50 µL de fluido oral. Assim sendo, este método representa uma excelente alternativa às técnicas convencionais na área da toxicologia e da monitorização clínica, permitindo melhorar a assistência ao doente, diminuir o risco de toxicidade, reduzir reações adversas e/ou interações farmacológicas como também a falha na adesão à terapêutica

Novel affinity chromatography processes for the purification of plasmid DNA using small aromatic molecules

Molecular therapies are gaining importance as an effective therapeutic approach for various types of diseases. The most efficient vectors used to introduce the therapeutic genes are of viral origin however, non-viral vectors based on pDNA are gaining popularity due to their superior safety and easy of production. These factors have increased the demand for high quantities of pharmaceutical grade plasmid DNA (pDNA). Therefore, the research for more efficient pDNA purification protocols has also increased. Moreover, the final pDNA product must meet stringent quality criteria established by the regulatory agencies. Liquid chromatography is the method of choice for the purification of pDNA, since it is simple, robust, versatile and high reproducible. The most important features of a chromatographic procedure are the use of suitable stationary phases and ligands. As conventional purification protocols are being replaced by more sophisticated and selective procedures, the focus changes towards designing and selecting ligands of high affinity and specificity. In fact, the chemical composition of the chromatographic supports determines the interactions established with the target molecules, allowing their preferential retention over the undesirable ones. With these facts in mind, the aim of this work was to develop new chromatographic methods for the purification of pharmaceutical grade pDNA, with the purpose of improving the overall procedures to more effective, simple, economic and environmental-friendly ones. The minor groove binder berenil and the intercalator 3,8-diamino-6-phenylphenanthridine (DAPP) where chosen to be used as ligands in pDNA chromatographic purification studies. They were immobilized to an epoxy-activated Sepharose matrix using a relatively mild curing method, without a catalyst and with quite small ligand:Sepharose weight ratios. Berenil binds pDNA preferentially through hydrophobic interactions but other types of interaction contributions cannot be neglected. It was shown to be quite effective at separating and purifying pDNA from clarified and non-clarified cell lysates, using three different approaches, although isoform resolution was not obtained in either case. Using mild amounts of ammonium sulphate in the eluent, berenil-Sepharose support was able to purify distinct pDNA of two different sizes from clarified cell lysates. Moreover, the ability was continual when the clarification process was replaced by a second chromatographic run. In all cases plasmid solutions were in accordance to the specifications of a pharmaceutical product, however the yields were quite different: two consecutive chromatographic runs lead to lower recoveries (33%) and smaller pDNA molecules have higher recoveries using one run through the column (85% vs 45%). A negative chromatography approach was also performed with berenil-Sepharose, showing some advantages in terms of salt usage as well as procedure time. In this case the recovery yield was quite good (87%) and although pDNA solutions had a comparable purity to that obtained with the other approaches, the gDNA reduction was not so effective. DAPP is slightly A-T specific and binds DNA through non-covalent, reversible stacking interactions of the condensed aromatic moiety into two successive base pairs, while the phenyl residue gets inserted into the minor groove. In addition, protonated DAPP molecules bind to DNA much strongly due to the generation of strong electrostatic interactions. Plasmid DNA binding to DAPP-Sepharose varies with pH and is affected by the presence of salt in the eluent. In fact, total retention of clarified lysate components was only possible with a pH below DAPP's free state pKa (5.8) and the presence of salt destabilizes that same retention. So, the elution of bound species was simply performed by adding small amounts of sodium chloride to the buffers. These features were successfully applied for purification of sc pDNA with two distinct sizes that were obtained according to the regulatory agencies specifications. Once more, the recovery yield of the smaller pDNA molecule was higher than the one obtained for the largest one (94% vs 65%). In conclusion, DAPP-Sepharose showed exceptional characteristics to be used as an affinity support for the purification of pharmaceutical grade sc pDNA. In comparison with berenil- Sepharose, it uses much smaller amounts of salt, with less economic and environmental impact, while improving the quality and yield of the obtained plasmid fractions. Moreover, it is able to separate sc pDNA from linear and oc isoforms even in complex lysates. Moreover, combining DAPP-Sepharose chromatography with other optimized production, extraction and clarification procedures, can offer a number of advantages for pharmaceutical pDNA purification. Also, since the most significant disadvantage of this DAPP-Sepharose support is the relatively low capacity for pDNA, which in turn is strongly related to the solid matrix used, other more stable stationary phases with low pressure drops and interconnected macropores, that allow a high mass transfer of solutes, are quite fascinating alternatives.Fundação para a Ciência e a Tecnologia (FCT

La dolce vita: el papel de los azúcares en la biosíntesis de glicoproteínas

El retículo endoplásmico es el lugar en donde se sintetizan las proteínas que ingresan a la vía secretoria. Previo a su salida, las proteínas adquieren su estructura terciaria y, de ser necesario, se ensamblan en oligómeros funcionales. Para facilitar estos procesos existen una gran variedad de chaperonas y enzimas facilitadoras del plegamiento. Asimismo, el estado de plegamiento es monitoreado por un sistema de control de calidad, el cual retiene en el retículo endoplásmico a aquellas especies que no han adquirido su conformación nativa. Si una proteína es incapaz de plegarse correctamente es retenida en el retículo endoplásmico y eventualmente es retrotranslocada al citosol para ser degradada por el proteasoma. Concomitantemente con el plegamiento tienen lugar diversas modificaciones postraduccionales, siendo las más destacadas la N-glicosilación y la formación de puentes disulfuro. Cerca de un cuarto de las proteínas de una célula eucarionte son N-glicosiladas, siendo la modificación postraduccional más frecuente. En las proteínas que alcanzaron su destino final los N-glicanos cumplen papeles fundamentales en diversos procesos de reconocimiento celular. Sin embargo los N-glicanos son utilizados también durante la maduración de las glicoproteínas como un sistema que codifica información acerca de su estado conformacional, siendo un elemento clave en varias instancias decisivas a lo largo de la vía secretoria. En este trabajo se presentan las diversas etapas que atraviesa una proteína desde su ingreso al retículo endoplásmico hasta su llegada a su destino final, poniendo especial atención a las funciones tempranas de los N-glicanos.Fil: Caramelo, Julio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

A novel plasmonic nanostructure for localized near-field light enhancement

The combination of plasmonic structures with dielectric materials, metallic oxides and in particular, with two dimensional (2D) materials has been a subject of great interest in the scientific community for a wide range of applications, such as optoelectronic devices, solar cells, or photochemistry. Plasmonic properties have the potential to enhance the capabilities of 2D materials for harvesting light, which alone have a low efficiency due to low absorption (approximately only 11%). This work presents a new plasmonic structure, “nanohippo” with a perspective to integrate a monolayer material inside the cavity, being this nanostructure directly excited by the incident light taking advantage of localized surface plasmonic resonance (LSPR). The samples were prepared by colloidal lithography and material deposition was made through an electron beam assisted evaporation system. A fabrication method was developed to create a cavity by using a sacrificial material and etching it later on. The sample’s characterization consisted in a morphologic analysis by Scanning Electron Microscopy (SEM), the optical response was studied both theoretically and experimentally by Finite-difference-time-domain (FDTD) as well as experimentally by spectrophotometry. Finally, an elemental analysis was performed via X-ray photoelectron microscopy (XPS). The diameter and height of the structures were studied (different sizes nanoparticles and thicker layers of bottom layer gold) reaching to a structure that presented a plasmonic cavity. This nanostructure, with a new geometry, presented a visible plasmonic nanocavity with up to sixty times more enhancement of the electrical field inside it

Analysis of Donald Trump’s emotional speech on the 2016 election campaign

Las emociones están presentes en nuestras relaciones personales, sociales y profesionales, repercuten en la manera de interactuar con los demás, en cómo éstos nos perciben y cómo nosotros los percibimos. Por ello, es importante a la hora de comunicar dominar una serie de habilidades sociales y comunicativas para que la comunicación sea efectiva y el mensaje sea percibido de la manera deseada. Sin embargo, las emociones y su correcta expresión dependerán de los objetivos que cada político quiera alcanzar. Por ello, la comunicación emocional irá siempre vinculada al contexto político y social y a las emociones de la ciudadanía con el objetivo de explotarlas en el discurso político a través la comunicación verbal y no verbal. En primer lugar, abordamos el tema de las emociones y el papel que juegan en el discurso político, en la persuasión de la ciudadanía y en su comportamiento. Debido a la importancia que tiene en cuanto a comunicación emocional, hemos estudiado la comunicación no verbal como principal herramienta de comunicación afectiva. Posteriormente, analizamos la comunicación no verbal de Donald Trump en diferentes declaraciones institucionales, con el fin de demostrar que la comunicación política y emocional varía en función del contexto y de los objetivos del líder político. Por último, y tras un profundo análisis del discurso, tratamos de poner de manifiesto y justificar la influencia de las emociones en la comunicación política y cómo éstas son determinantes en la percepción del mensaje.Emotions are present in our personal, social and professional relationships, this, affecting the way we interact with others, about how they perceive us and how we perceive them. Therefore, it is important when communicating with others, to master a series of socialand communication skills so that communication is effective and the message is perceived in the desired way. However, the emotions and their correct expression will depended on the objectives that each politician wants to achieve. Therefore, emotional communication will always be linked to the political and social context and to the emotions of the citizenry with the aim of exploiting them in the political discourse through verbal and non-verbal communication. First of all, we address the subject of emotions and the role they play in the political discourse, in the persuasion of the citizens and their behaviour. Due to its importance in terms of emotional communication, we have studied non-verbal communication as the main tool of effective communication. Subsequently, we analyzed Donald Trump’s nonverbal communication in different institutional statements, in order to demonstrate that political and emotional communication varies depending the context and the objectives of the political leader. Finally, after a thorough analysis of the discourse, we try to highlight and justify the influence of emotions in political communication and how these are determinant in the perception of the message