Microfluidic platforms for the characterization of in meso membrane protein crystallization

Membrane proteins, which reside in the membranes of cells, play a critical role in many important biological processes including cellular signaling, immune response, and material and energy transduction. Because of their key role in maintaining the environment within cells and facilitating intercellular interactions, understanding the function of these proteins is of tremendous medical and biochemical significance. Indeed, the malfunction of membrane proteins has been linked to numerous diseases including diabetes, cirrhosis of the liver, cystic fibrosis, cancer, Alzheimer's disease, hypertension, epilepsy, cataracts, tubulopathy, leukodystrophy, Leigh syndrome, anemia, sensorineural deafness, and hypertrophic cardiomyopathy.1-3 However, the structure of many of these proteins and the changes in their structure that lead to disease-related malfunctions are not well understood. Additionally, at least 60% of the pharmaceuticals currently available are thought to target membrane proteins, despite the fact that their exact mode of operation is not known.4-6 Developing a detailed understanding of the function of a protein is achieved by coupling biochemical experiments with knowledge of the structure of the protein. Currently the most common method for obtaining three-dimensional structure information is X-ray crystallography. However, no a priori methods are currently available to predict crystallization conditions for a given protein.7-14 This limitation is currently overcome by screening a large number of possible combinations of precipitants, buffer, salt, and pH conditions to identify conditions that are conducive to crystal nucleation and growth.7,9,11,15-24 Unfortunately, these screening efforts are often limited by difficulties associated with quantity and purity of available protein samples. While the two most significant bottlenecks for protein structure determination in general are the (i) obtaining sufficient quantities of high quality protein samples and (ii) growing high quality protein crystals that are suitable for X-ray structure determination,7,20,21,23,25-47 membrane proteins present additional challenges. For crystallization it is necessary to extract the membrane proteins from the cellular membrane. However, this process often leads to denaturation. In fact, membrane proteins have proven to be so difficult to crystallize that of the more than 66,000 structures deposited in the Protein Data Bank,48 less than 1% are for membrane proteins, with even fewer present at high resolution (< 2Å)4,6,49 and only a handful are human membrane proteins.49 A variety of strategies including detergent solubilization50-53 and the use of artificial membrane-like environments have been developed to circumvent this challenge.43,53-55 In recent years, the use of a lipidic mesophase as a medium for crystallizing membrane proteins has been demonstrated to increase success for a wide range of membrane proteins, including human receptor proteins.54,56-62 This in meso method for membrane protein crystallization, however, is still by no means routine due to challenges related to sample preparation at sub-microliter volumes and to crystal harvesting and X-ray data collection. This dissertation presents various aspects of the development of a microfluidic platform to enable high throughput in meso membrane protein crystallization at a level beyond the capabilities of current technologies. Microfluidic platforms for protein crystallization and other lab-on-a-chip applications have been well demonstrated.9,63-66 These integrated chips provide fine control over transport phenomena and the ability to perform high throughput analyses via highly integrated fluid networks. However, the development of microfluidic platforms for in meso protein crystallization required the development of strategies to cope with extremely viscous and non-Newtonian fluids. A theoretical treatment of highly viscous fluids in microfluidic devices is presented in Chapter 3, followed by the application of these strategies for the development of a microfluidic mixer capable of preparing a mesophase sample for in meso crystallization at a scale of less than 20 nL in Chapter 4. This approach was validated with the successful on chip in meso crystallization of the membrane protein bacteriorhodopsin. In summary, this is the first report of a microfluidic platform capable of performing in meso crystallization on-chip, representing a 1000x reduction in the scale at which mesophase trials can be prepared. Once protein crystals have formed, they are typically harvested from the droplet they were grown in and mounted for crystallographic analysis. Despite the high throughput automation present in nearly all other aspects of protein structure determination, the harvesting and mounting of crystals is still largely a manual process. Furthermore, during mounting the fragile protein crystals can potentially be damaged, both from physical and environmental shock. To circumvent these challenges an X-ray transparent microfluidic device architecture was developed to couple the benefits of scale, integration, and precise fluid control with the ability to perform in situ X-ray analysis (Chapter 5). This approach was validated successfully by crystallization and subsequent on-chip analysis of the soluble proteins lysozyme, thaumatin, and ribonuclease A and will be extended to microfluidic platforms for in meso membrane protein crystallization. The ability to perform in situ X-ray analysis was shown to provide extremely high quality diffraction data, in part as a result of not being affected by damage due to physical handling of the crystals. As part of the work described in this thesis, a variety of data collection strategies for in situ data analysis were also tested, including merging of small slices of data from a large number of crystals grown on a single chip, to allow for diffraction analysis at biologically relevant temperatures. While such strategies have been applied previously,57,59,61,67 they are potentially challenging when applied via traditional methods due to the need to grow and then mount a large number of crystals with minimal crystal-to-crystal variability. The integrated nature of microfluidic platforms easily enables the generation of a large number of reproducible crystallization trials. This, coupled with in situ analysis capabilities has the potential of being able to acquire high resolution structural data of proteins at biologically relevant conditions for which only small crystals, or crystals which are adversely affected by standard cryocooling techniques, could be obtained (Chapters 5 and 6). While the main focus of protein crystallography is to obtain three-dimensional protein structures, the results of typical experiments provide only a static picture of the protein. The use of polychromatic or Laue X-ray diffraction methods enables the collection of time resolved structural information. These experiments are very sensitive to crystal quality, however, and often suffer from severe radiation damage due to the intense polychromatic X-ray beams. Here, as before, the ability to perform in situ X-ray analysis on many small protein crystals within a microfluidic crystallization platform has the potential to overcome these challenges. An automated method for collecting a "single-shot" of data from a large number of crystals was developed in collaboration with the BioCARS team at the Advanced Photon Source at Argonne National Laboratory (Chapter 6). The work described in this thesis shows that, even more so than for traditional structure determination efforts, the ability to grow and analyze a large number of high quality crystals is critical to enable time resolved structural studies of novel proteins. In addition to enabling X-ray crystallography experiments, the development of X-ray transparent microfluidic platforms also has tremendous potential to answer other scientific questions, such as unraveling the mechanism of in meso crystallization. For instance, the lipidic mesophases utilized during in meso membrane protein crystallization can be characterized by small angle X-ray diffraction analysis. Coupling in situ analysis with microfluidic platforms capable of preparing these difficult mesophase samples at very small volumes has tremendous potential to enable the high throughput analysis of these systems on a scale that is not reasonably achievable using conventional sample preparation strategies (Chapter 7). In collaboration with the LS-CAT team at the Advanced Photon Source, an experimental station for small angle X-ray analysis coupled with the high quality visualization capabilities needed to target specific microfluidic samples on a highly integrated chip is under development. Characterizing the phase behavior of these mesophase systems and the effects of various additives present in crystallization trials is key for developing an understanding of how in meso crystallization occurs. A long term goal of these studies is to enable the rational design of in meso crystallization experiments so as to avoid or limit the need for high throughput screening efforts. In summary, this thesis describes the development of microfluidic platforms for protein crystallization with in situ analysis capabilities. Coupling the ability to perform in situ analysis with the small scale, fine control, and the high throughput nature of microfluidic platforms has tremendous potential to enable a new generation of crystallographic studies and facilitate the structure determination of important biological targets. The development of platforms for in meso membrane protein crystallization is particularly significant because they enable the preparation of highly viscous mixtures at a previously unachievable scale. Work in these areas is ongoing and has tremendous potential to improve not only current the methods of protein crystallization and crystallography, but also to enhance our knowledge of the structure and function of proteins which could have a significant scientific and medical impact on society as a whole. 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Evaluation of the Genotoxicity of Chitosan Nanoparticles for Use in Food Packaging Films

The use of nanoparticles in food packaging has been proposed on the basis that it could improve protection of foods by, for example, reducing permeation of gases, minimizing odor loss, and increasing mechanical strength and thermal stability. Consequently, the impacts of such nanoparticles on organisms and on the environment need to be investigated to ensure their safe use. In an earlier study, Moura and others (2008a) described the effect of addition of chitosan (CS) and poly(methacrylic acid) (PMAA) nanoparticles on the mechanical properties, water vapor, and oxygen permeability of hydroxypropyl methylcellulose films used in food packaging. Here, the genotoxicity of different polymeric CS/PMAA nanoparticles (size 60, 82, and 111 nm) was evaluated at different concentration levels, using the Allium cepa chromosome damage test as well as cytogenetic tests employing human lymphocyte cultures. Test substrates were exposed to solutions containing nanoparticles at polymer mass concentrations of 1.8, 18, and 180 mg/L. Results showed no evidence of DNA damage caused by the nanoparticles (no significant numerical or structural changes were observed), however the 82 and 111 nm nanoparticles reduced mitotic index values at the highest concentration tested (180 mg/L), indicating that the nanoparticles were toxic to the cells used at this concentration. In the case of the 60 nm CS/PMAA nanoparticles, no significant changes in the mitotic index were observed at the concentration levels tested, indicating that these particles were not toxic. The techniques used show promising potential for application in tests of nanoparticle safety envisaging the future use of these materials in food packaging.

MEDIDAS DE NÚCLEO INFLACIONARIO PARA COSTA RICA

En este documento se elaboran y evalúan medidas alternativas de núcleo inflacionario para Costa Rica. La idea fundamental contempla al núcleo inflacionario como un indicador de la tendencia subyacente de la inflación capaz de capturar el componente del cambio total de precios común a todos los bienes y servicios, cuya persistencia se mantendría en el mediano y largo plazo y que excluye los cambios en los precios relativos de estos. La medida de núcleo inflacionario seleccionada se contrasta con el Índice de Núcleo Inflacionario (INI), indicador de inflación subyacente actualmente utilizado por el Banco Central de Costa Rica (BCCR).El Índice Subyacente de Inflación (ISI), definido como una medida de núcleo inflacionario que excluye un 30,7% del peso total del Índice de Precios al Consumidor (IPC), refleja más fielmente la tendencia subyacente de la inflación y logra capturar el movimiento más permanente del nivel general de precios, aislando las variaciones en precios relativos. Además, el ISI es fácil de calcular e interpretar lo cual ayuda a incrementar la transparencia y credibilidad de la política monetaria. También es un indicador oportuno, aumentando su valor para los que formulan la política monetaria. Por último, el ISI supera algunas de las limitaciones del INI, como son la falta de un criterio estadístico para definir el punto de corte de los bienes y servicios a excluir y el alto porcentaje del peso total del IPC eliminado. AbstractThis paper builds and evaluates several alternative measures of core inflation for Costa Rica. The chosen measure of core inflation is contrasted with the core inflation index (INI), which is the indicator of underlying inflation used today by the Central Bank of Costa Rica (BCCR). The main idea is that core inflation is a good indicator of the underlying inflation and catches the part of overall price change common to all the goods and services that is expected to persist in the medium-term and long-term, and excludes changes in the relative prices of goods and services.The Underlying Inflation Index (ISI) is defined as a measure of core inflation which excludes 30,7% of the total weight of the Consumer Price Index (IPC), it is the most closely related with inflation´s underlying trend and catches the component of overall price change that is expected to persist in the general level of prices. Furthermore, the ISI is easy to compute and to follow, increasing the transparency and credibility of monetary policy and moreover is an timely indicator increasing its value for the monetary policy makers. Finally, the ISI exceed some limitations of INI, as the absence of statistic criterion to define the cutting point of goods and services to exclude and the high percent of total weight eliminated of IPC.

Imagen universal del hombre latinoamericano

En este artículo se presenta un análisis de la imagen del hombre de América Latina enfatizando que no es un "buen salvaje", sino, un hombre lleno de soledad, de muerte, de injusticia, de dolor, de goce de vivir, metido en la crisis moral, económica y política del mundo.

The Amplifier - v. 11, no. 7

In this issue...KXLF TV, Mine Rescue, Talent Show, Linda Plubell, Gulf Oil Company, Student Loans, Theta Tau, Alaska, Newman Club

#11 ATGC

Combined media monoprints on photographic paper. 8" x 10", DNA/Recombination Series. The Museum of Contemporary Photography, Chicago

Mediação civil e comercial na União Europeia

Na sua génese mais purista, a mediação é um processo voluntário, flexível e informal em que um terceiro neutro e imparcial auxilia as partes litigantes a encontrar por si mesmas uma solução mutuamente satisfatória para o seu litígio. As Instituições Europeias têm estado atentas à potencialidade da mediação que obtém soluções amigáveis e promove a paz social para além de servir de ferramenta para o descongestionamento dos tribunais judiciais contribuindo para a melhoria do acesso à justiça. A Diretiva 2008/52/CE pretende harmonizar as legislações do Estados-Membros relativamente à mediação em matéria civil e comercial e apresenta uma postura flexível quanto à introdução de elementos de mediação obrigatórios desde que tal não impeça o acesso à justiça. Há autores que defendem que a mediação obrigatória desvirtua a sua essência voluntária transformando-a num mero expediente dilatório e outros entendem que a obrigatoriedade gera hábitos e cultura de mediação. Portugal e Espanha regulamentaram a mediação voluntária, por outro lado Itália introduziu a mediação obrigatória como condição de procedibilidade da ação judicial em determinadas matérias, para promover a mediação e aliviar a sobrecarga dos tribunais judiciais. O estudo “Rebooting”, publicado pelo Parlamento, apresenta resultados dececionantes uma vez que o número de mediações nos Estados-Membros corresponde a menos de 1% dos casos litigados na União Europeia, além disso a maior partes dos especialistas inquiridos entendeu que só a introdução de medidas que incluam algum grau de compulsoriedade é capaz de aumentar o número de casos mediados. A Comissão Europeia para a Eficiência da Justiça indica que os tribunais portugueses de 1ª instância têm sérias dificuldades em dar resposta aos processos pendentes e aos que dão entrada. O legislador português deveria considerar a introdução da mediação obrigatória com opt-out de forma a promover a mediação e agilizar o sistema judicial.

Revisión del uso de los medicamentos biosimilares vs. biológicos: implicaciones para la salud en Colombia.

Este documento tiene como objetivo describir las implicaciones para la salud con el uso de medicamentos biosimilares en comparación con los medicamentos biológicos en Colombia. Así mismo, describir el contexto normativo acerca del uso de medicamentos biosimilares, las recomendaciones y lineamientos sobre seguridad y efectividad del uso de medicamentos Biosimilares y Biológicos, partiendo de sus diferencias biomoleculares. Para esto, se desarrolló una revisión documental electrónica y manual de la literatura en bases de datos, revistas y libros limitada a términos MeSH. La selección de los artículos incluyo documentos completos publicados en revistas indexadas de los últimos 10 años, en español e inglés; la información recolectada se organizó para la construcción del presente documento. Concluyendo, se encontró que las patentes de muchos medicamentos biológicos han vencido o están próximas a caducar y varios biosimilares están desarrollándose y comercializándose incluso en países sin regulaciones estrictas. Los biosimilares nunca podrán ser iguales al original por su complejidad molecular, por ello debemos integrarlos a los sistemas de farmacovigilancia mejorando trazabilidad e identificando su origen mientras se establecen denominaciones comunes distinguibles. La evidencia actual sugiere que la regulación de medicamentos biosimilares debe ser evaluada y armonizada en todo el mundo.

Asociación entre la escala de riesgo Framingham y el puntaje de calcio coronario

Introducción: Entre las diferentes herramientas clínicas para evaluar la presencia de enfermedad coronaria mediante puntajes, la más usada es la Escala de Riesgo cardiovascular de Framingham. Desde hace unos años, se creó el puntaje de calcio coronario el cual mide el riesgo cardiovascular según la presencia de placas ateromatosas vistas por tomografía computarizada. Se evaluó la asociación entre la escala de Framigham y el puntaje de calcio coronario en una población de sujetos sanos asintomáticos. Metodología: Se realizó un estudio transversal para evaluar la asociación entre el puntaje de calcio coronario y la escala de Framingham en sujetos asintomáticos que se practicaron exámen médico preventivo en la Fundación Cardioinfantil- Instituto de Cardiología (FCI-IC) en el periodo comprendido entre 1 de Julio 2011 hasta el 31 de octubre de 2015. Resultados: Se evaluaron 262 pacientes en total. La prevalencia de riesgo cardiovascular fue bajo en un 77.86% de la población, medio en 18.70% y alto en 3.44%, según la escala de Framingham. El riesgo cardiovascular según el puntaje de Calcio coronario fue nulo 70.99%, bajo en 21.75%, medio en 4.19%, severo en 3.05%. Se encontró una asociación entre ambos puntajes para riesgo estadísticamente significativa (p0,00001) Discusión: El riesgo cardiovascular establecido por escala de Framingham se relaciona de forma significativa con la presencia de placas aterioscleróticas. El estudio demostró que en una muestra de sujetos asintomáticos, hay una alteración estructural coronaria temprana.

Estado de las enfermedades raras que cursan con discapacidad intelectual en Colombia

Las enfermedades raras o huérfano son una problemática que ha tomado mucha importancia en el contexto mundial del presente siglo, estas se han definido como crónicas, de difícil tratamiento de sus síntomas y con baja prevalencia en la población; muchas de estas enfermedades cursan con varios tipos de discapacidad, siendo el objetivo del presente trabajo el enfocarse en aquellas enfermedades raras que cursan con discapacidad intelectual. Para poder profundizar en estas enfermedades se realizó una revisión teórica sobre las enfermedades raras, así como de la discapacidad psíquica y su importancia a nivel mundial y nacional. A partir de estas definiciones, se revisaron en profundidad 3 enfermedades raras que cursan con discapacidad intelectual en el contexto colombiano, como son: el síndrome de Rett, el síndrome de Prader-Willi y el síndrome de X frágil. En cada una de estas enfermedades además se explicaron los tipos de diagnóstico, intervención, prevención, grupos de apoyo y tipos de evaluación que más se usan en el contexto nacional

Determinação de antocianinas e compostos fenólicos totais em diferentes genótipos de açaí.

O açaizeiro é uma palmeira nativa da região Amazônica que alcançou um enorme potencial de mercado em função de suas características funcionais. A Embrapa Amazônia Oriental possui um Banco Ativo de Germoplasma (BAG) de açaí e o estudo da composição de seus frutos é indispensável para a tomada de decisão sobre genótipos a fim de identificar aqueles que possuem características superiores. Assim, o objetivo desta pesquisa foi determinar compostos bioativos em genótipos deste BAG e no presente trabalho apresentam-se os resultados referentes a dez genótipos. Observou-se uma variação de 309,17 a 1341,04 mg/100g e 254,43 a 1147,64 mg/100g para os teores de antocianinas totais e monoméricas, respectivamente. Nestes pigmentos o destaque foi para o genótipo L7PL11. Já para compostos fenólicos totais, o destaque foi para o genótipo L12PL20, com um teor médio de 2107,68 mg/100g. De maneira geral, observou-se diferença significativa na maioria dos genótipos estudados, de acordo com o teste estatístico aplicado