Switchable lanthanide fluorescence probes in homogenous molecular diagnostics

The number of molecular diagnostic assays has increased tremendously in recent years.Nucleic acid diagnostic assays have been developed, especially for the detection of human pathogenic microbes and genetic markers predisposing to certain diseases. Closed-tube methods are preferred because they are usually faster and easier to perform than heterogenous methods and in addition, target nucleic acids are commonly amplified leading to risk of contamination of the following reactions by the amplification product if the reactions are opened. The present study introduces a new closed-tube switchable complementation probes based PCR assay concept where two non-fluorescent probes form a fluorescent lanthanide chelate complex in the presence of the target DNA. In this dual-probe PCR assay method one oligonucleotide probe carries a non-fluorescent lanthanide chelate and another probe a light absorbing antenna ligand. The fluorescent lanthanide chelate complex is formed only when the non-fluorescent probes are hybridized to adjacent positions into the target DNA bringing the reporter moieties in close proximity. The complex is formed by self-assembled lanthanide chelate complementation where the antenna ligand is coordinated to the lanthanide ion captured in the chelate. The complementation probes based assays with time-resolved fluorescence measurement showed low background signal level and hence, relatively high nucleic acid detection sensitivity (low picomolar target concentration). Different lanthanide chelate structures were explored and a new cyclic seven dentate lanthanide chelate was found suitable for complementation probe method. It was also found to resist relatively high PCR reaction temperatures, which was essential for the PCR assay applications. A seven-dentate chelate with two unoccupied coordination sites must be used instead of a more stable eight- or nine-dentate chelate because the antenna ligand needs to be coordinated to the free coordination sites of the lanthanide ion. The previously used linear seven-dentate lanthanide chelate was found to be unstable in PCR conditions and hence, the new cyclic chelate was needed. The complementation probe PCR assay method showed high signal-to-background ratio up to 300 due to a low background fluorescence level and the results (threshold cycles) in real-time PCR were reached approximately 6 amplification cycles earlier compared to the commonly used FRET-based closed-tube PCR method. The suitability of the complementation probe method for different nucleic acid assay applications was studied. 1) A duplex complementation probe C. trachomatis PCR assay with a simple 10-minute urine sample preparation was developed to study suitability of the method for clinical diagnostics. The performance of the C. trachomatis assay was equal to the commercial C. trachomatis nucleic acid amplification assay containing more complex sample preparation based on DNA extraction. 2) A PCR assay for the detection of HLA-DQA1*05 allele, that is used to predict the risk of type 1 diabetes, was developed to study the performance of the method in genotyping. A simple blood sample preparation was used where the nucleic acids were released from dried blood sample punches using high temperature and alkaline reaction conditions. The complementation probe HLA-DQA1*05 PCR assay showed good genotyping performance correlating 100% with the routinely used heterogenous reference assay. 3) To study the suitability of the complementation probe method for direct measurement of the target organism, e.g., in the culture media, the complementation probes were applied to amplificationfree closed-tube bacteriophage quantification by measuring M13 bacteriophage ssDNA. A low picomolar bacteriophage concentration was detected in a rapid 20- minute assay. The assay provides a quick and reliable alternative to the commonly used and relatively unreliable UV-photometry and time-consuming culture based bacteriophage detection methods and indicates that the method could also be used for direct measurement of other micro-organisms. The complementation probe PCR method has a low background signal level leading to a high signal-to-background ratio and relatively sensitive nucleic acid detection. The method is compatible with simple sample preparation and it was shown to tolerate residues of urine, blood, bacteria and bacterial culture media. The common trend in nucleic acid diagnostics is to create easy-to-use assays suitable for rapid near patient analysis. The complementation probe PCR assays with a brief sample preparation should be relatively easy to automate and hence, would allow the development of highperformance nucleic acid amplification assays with a short overall assay time.

Micro Data Repositories: Increasing the Value of Research on the Web

Presentation at Open Repositories 2014, Helsinki, Finland, June 9-13, 2014

The role of micro-ribonucleic acids in normal hematopoiesis and leukemic T-lymphogenesis

Micro-ribonucleic acids (microRNAs) are small molecules containing 20-23 nucleotides. Despite their small size, it is likely that almost every cellular process is regulated by them. Moreover, aberrant microRNA expression has been involved in the development of various diseases, including cancer. Although many data are available about the role of microRNAs in various lymphoproliferative disorders, their impact on the development of acute lymphoblastic leukemia of T-cell progenitors is largely unknown. In this review, we present recent information about how specific microRNAs are expressed and regulated during malignant T-lymphopoiesis and about their role during normal hematopoiesis.

Plasma concentrations and placental immunostaining of interleukin-10 and tumornecrosis factor-α as predictors of alterations in the embryo-fetal organism and the placental development of diabetic rats

Interleukin-10 (IL-10) appears to be the key cytokine for the maintenance of pregnancy and inhibits the secretion of inflammatory cytokines such as tumor necrosis factor-α (TNF-α). However, there are no studies evaluating the profile of these cytokines in diabetic rat models. Thus, our aim was to analyze IL-10 and TNF-α immunostaining in placental tissue and their respective concentrations in maternal plasma during pregnancy in diabetic rats in order to determine whether these cytokines can be used as predictors of alterations in the embryo-fetal organism and in placental development. These parameters were evaluated in non-diabetic (control; N = 15) and Wistar rats with streptozotocin (STZ)-induced diabetes (N = 15). At term, the dams (100 days of life) were killed under anesthesia and plasma and placental samples were collected for IL-10 and TNF-α determinations by ELISA and immunohistochemistry, respectively. The reproductive performance was analyzed. Plasma IL-10 concentrations were reduced in STZ rats compared to controls (7.6 ± 4.5 vs 20.9 ± 8.1 pg/mL). The placental scores of immunostaining intensity did not differ between groups (P > 0.05). Prevalence analysis showed that the IL-10 expression followed TNF-α expression, showing a balance between them. STZ rats also presented impaired reproductive performance and reduced plasma IL-10 levels related to damage during early embryonic development. However, the increased placental IL-10 as a compensatory mechanism for the deficit of maternal regulation permitted embryo development. Therefore, the data suggest that IL-10 can be used as a predictor of changes in the embryo-fetal organism and in placental development in pregnant diabetic rats.

Improved biological performance of magnesium by micro-arc oxidation

Magnesium and its alloys have recently been used in the development of lightweight, biodegradable implant materials. However, the corrosion properties of magnesium limit its clinical application. The purpose of this study was to comprehensively evaluate the degradation behavior and biomechanical properties of magnesium materials treated with micro-arc oxidation (MAO), which is a new promising surface treatment for developing corrosion resistance in magnesium, and to provide a theoretical basis for its further optimization and clinical application. The degradation behavior of MAO-treated magnesium was studied systematically by immersion and electrochemical tests, and its biomechanical performance when exposed to simulated body fluids was evaluated by tensile tests. In addition, the cell toxicity of MAO-treated magnesium samples during the corrosion process was evaluated, and its biocompatibility was investigated under in vivo conditions. The results of this study showed that the oxide coating layers could elevate the corrosion potential of magnesium and reduce its degradation rate. In addition, the MAO-coated sample showed no cytotoxicity and more new bone was formed around it during in vivo degradation. MAO treatment could effectively enhance the corrosion resistance of the magnesium specimen and help to keep its original mechanical properties. The MAO-coated magnesium material had good cytocompatibility and biocompatibility. This technique has an advantage for developing novel implant materials and may potentially be used for future clinical applications.

Reconocimiento de alimentos vegetales: caracterización micro-gráfica del grano de avena

Se estudia la estructura micrográfica del grano de seis variedades de avena con la finalidad de su caracterización, para desarrollar parámetros de identificación en alimentos elaborados con la misma y, consecuentemente, determinar su autenticidad, contribuyendo a optimizar la producción, la comercialización y el consumo del cereal y sus derivados. El diseño experimental consistió en el estudio micrográfico de los granos vestidos y desnudos efectuando un análisis morfológico mediante observación con lupa binocular y fotografía, ultraestructural utilizando microscopio electrónico de barrido, micrográfico y micrométrico, empleando el sistema de video microscopia digitalizado y software adecuado. Dada su variabilidad natural, los estudios se efectuaron durante tres temporadas consecutivas sobre muestras cosechadas de variedades procedentes de cultivos de semillas certificadas, y sobre alimentos procesados (avena arrollada y salvado de avena comerciales). Los resultados consistieron en diseños micrográficos, y en valores micrométricos de gránulos de almidón relacionados, además, en modelos matemáticos. En todos los casos se validó estadísticamente. Como parámetros micrográficos de caracterización se seleccionaron las estructuras diferenciales, que revelaron una presencia constante en el vegetal y resistieron los tratamientos tecnológicos, y las características y dimensiones del almidón.

Tratamento térmico do amido de batata-doce (Ipomoea batatas L.) sob baixa umidade em micro-ondas

O objetivo deste trabalho foi avaliar o efeito do tratamento térmico sob baixa umidade (TTBU) aplicado por forno micro-ondas sobre as propriedades estruturais e funcionais do amido de batata-doce e compará-las com as propriedades de amido tratado pelo método convencional. O amido extraído dessa raiz foi submetido à modificação física, nas umidades de 25 e 35%, em forno convencional (90 °C/16 horas) e em microondas (35 a 90 °C/1 hora). O tratamento térmico sob baixa umidade resultou em alterações significativas no teor de amilose e em características como a cristalinidade, suscetibilidade enzimática, fator de expansão e propriedades de pasta. Tais variações evidenciam modificações na estrutura granular interna dos amidos, tanto em áreas cristalinas como amorfas do grânulo. As alterações conferidas pelo TTBU foram variáveis com o tipo de tratamento térmico e com o teor de umidade. A umidade das amostras também foi determinante na modificação da maioria das características do amido, como maior digestibilidade enzimática e redução da expansão, menores picos de viscosidade e quebras de viscosidade, independentemente do tipo de tratamento térmico aplicado. Considerando-se o tipo e a intensidade da modificação física do amido tratado pelo método convencional como referência, a utilização da energia de micro-ondas para esse mesmo fim precisa ser melhor estudada.

Head Space Solid Phase Micro-Extraction (HS - SPME) of volatile organic compounds produced by Sporidiobolus salmonicolor (CBS 2636)

The aim of the present study was the assessment of volatile organic compounds produced by Sporidiobolus salmonicolor (CBS 2636) using methyl and ethyl ricinoleate, ricinoleic acid and castor oil as precursors. The analysis of the volatile organic compounds was carried out using Head Space Solid Phase Micro-Extraction (HS - SPME). Factorial experimental design was used for investigating extraction conditions, verifying stirring rate (0-400 rpm), temperature (25-60 ºC), extraction time (10-30 minutes), and sample volume (2-3 mL). The identification of volatile organic compounds was carried out by Gas Chromatography with Mass Spectrum Detector (GC/MSD). The conditions that resulted in maximum extraction were: 60 ºC, 10 minutes extraction, no stirring, sample volume of 2.0 mL, and addition of saturated KCl (1:10 v/v). In the bio-production of volatile organic compounds the effect of stirring rate (120-200 rpm), temperature (23-33 ºC), pH (4.0-8.0), precursor concentration (0.02-0.1%), mannitol (0-6%), and asparagine concentration (0-0.2%) was investigated. The bio-production at 28 ºC, 160 rpm, pH 6,0 and with the addition of 0.02% ricinoleic acid to the medium yielded the highest production of VOCs, identified as 1,4-butanediol, 1,2,2-trimethylciclopropilamine, beta-ionone; 2,3-butanodione, pentanal, tetradecane, 2-isononenal, 4-octen-3-one, propanoic acid, and octadecane.

Desarrollo de un curso de micro controladores que incluye : el plan de estudio, apuntes y lista de prácticas de laboratorio [por] René Briones Lara

Tesis (Maestría en Ciencias de Ingeniería Eléctrica, con especialidad en Electrónica) U.A.N.L.

Modelo de exportación aplicable al comercio exterior, entre México y Guatemala, con beneficios sustanciales para la micro, pequeña y mediana empresa por Gabriel Esquinca Moreno

Tesis (Maestría en Administración Pública) U.A.N.L.

Estrategias para la creación, desarrollo y crecimiento de la micro y pequeña empresa

Tesis (Maestro en Ciencias de la Administración con Especialidad en Producción y Calidad) UANL, 2000

Los proyectos de inversión de la micro empresa por María del Rocío Aguilar Zamarripa

Tesis (Maestría en Contaduría Pública con Especialidad en Finanzas) U.A.N.L.

Gestión de la administración estratégica en la micro y pequeña empresa. Caso de estudio : Grupo Apolo Sistemas, S.A. de C.V.

Tesis (Maestría en Ciencias de la Administración con Especialidad en Relaciones Industriales) UANL, 2011.

Estructura organizacional como base para el éxito integral de micro, pequeñas y medianas empresas.

Tesis (Maestría en Psicología con Orientación Laboral y Organizacional) UANL, 2013