Encapsulación de células endometriales para el co-cultivo de embriones

Motivación: En la fecundación in vitro (FIV) se transfiere el embrión el dia 5, en el estadío de mórula-blastocito, para asegurar que se desarrolle correctamente y que haya menor probabilidad de aneuploidias. En pacientes con fallo de implantación, se desarrollo un método seguro, ético y efectivo para el co-cultivo con células epiteliales de endometrio (Simón y cols., 1999). Estos se siguen realizando de rutina en muchas clínicas, pero presentan una serie de desventajas como las contaminaciones bacterianas o fúngicas y la imposibilidad de llevarse a cabo en laboratorios externos que dificultan su trabajo. Este proyecto esta encaminado a implantar un nuevo método en las clínicas por sus ventajas: mejora de la calidad embrionaria, ahorro de tiempo en los laboratorios del FIV, mejora de la calidad del co-cultivo evitando posibles contaminaciones y un abaratamiento del sistema de co-cultivo al no tener que realizar el cultivo en los laboratorios FIV. Métodos: Se toman pequeñas biopsias endometriales mediante una fina cánula procedentes de donantes en los laboratorios FIV. Tras su obtención, se sigue un protocolo para la separación de epitelio y estroma (Simón y cols., 1999), en las que la mitad de las células epiteliales son encapsuladas en alginato cálcico y la otra mitad son sembradas en monocapa. Actualmente, estamos recogiendo los medios de cultivo tanto de las células encapsuladas como de las células en monocapa, los días 2, 4, 8 y 10 tras alcanzar la confluencia una vez han sido sembradas después de la encapsulación. Se analizarán en estos medios la presencia de factores que previamente han sido descritos en medios de cultivo de células epiteliales endometriales como IL-6. Se realizarán ensayos de ELISA para las proteínas secretadas y de Western para las células recogidas. Se correlacionarán los niveles de proteínas existentes en el medio con los medios de cultivo de las células en monocapa. Conclusiones: Tras la obtención y procesamiento de biopsias como se indico anteriormente, se demostró la viabilidad de las células endometriales en capsulas de alginato cálcico. Este sistema ofrece la posibilidad de desarrollar un nuevo método de implantación en las clínicas FIV mejorando los sistemas de co-cultivo actuales

Búsqueda de marcadores de embarazo ectópico en sangre periférica

Motivación: El embarazo ectópico es una anomalía en la implantación embrionaria y complicación ginecológica grave que afecta a más de un 1% de los embarazos naturales y hasta un 8% de las embarazadas en reproducción asistida, según las fuentes. Los métodos diagnósticos de embarazo ectópico a día de hoy se basan exclusivamente en métodos ecográficos y en medidas seriadas de B-hCG métodos que, en muchas ocasiones, se prolongan por una semana o más, para llegar a un diagnóstico fiable.En el Reino Unido el diagnóstico del embarazo ectópico genera un gasto anual de 12 millones de euros², datos extrapolables al resto de Europa. La existencia de un buen marcador de embarazo ectópico sustituiría la realización del test diario de β-hCG, siendo el mercado potencial de unos 13 millones de análisis por año.Métodos: Se recogieron seis muestras de sangre procedentes de mujeres con embarazo ectópico y otras seis de mujeres con embarazo intrauterino con edades gestacionales similares (entre 7 y 10 semanas) se almacenaron a -20ºC en tubos PAXGene hasta su posterior análisis. La extracción del RNA se realizó utilizando el método del Trizol (Sigma-Aldrich). Todas las muestras fueron hibridadas utilizando el “Whole Human Genome Oligo Microarray” (Agilent Technologies) que contiene mas de 44.000 sondas para el genoma humano. El microarray hibridado se escaneo mediante el escaner Axon 4100 (Molecular Devices) y los datos se extrajeron con el software Genepix 6.0 (Molecular Devices). El análisis de datos se llevo acabo con la plataforma GEPAS. Resultados: Se utilizaron seis muestras de embarazo ectópico y seis muestras de embarazo intrauterino para el análisis de microarray. Se generó una lista de 17 potenciales marcadores de embarazo ectópico con un fold change >4 o <-4 y con un p valor <0.05. De estos 17 genes se han validados por PCR cuantitativa los siguientes: ORM1, ORM2, LAMP3 y UTS2 utilizando nuevas muestras de embarazo ectópico (n=8) y de embarazo intrauterino (n=7). Después de descartar los valores atípicos se confirmaron algunos resultados del microarray.Conclusiones: Los resultados del microarray mostraron un total de 17 genes desregulados entre embarazos ectópicos e intrauterinos, concretamente 10 se sobre expresaban y 7 de ellos disminuían su expresión. Los genes ORM1, ORM2, LAMP3 y UTS2 se comprobaron por qPCR y mostraron diferencias significativas. . Al final de este estudio se comprobaran el resto de genes y se podrá diseñar un test de embarazo ectópico

A review of exposure assessment methods for epidemiological studies of health effects related to industrially contaminated sites

BACKGROUND: this paper is based upon work from COST Action ICSHNet. Health risks related to living close to industrially contaminated sites (ICSs) are a public concern. Toxicology-based risk assessment of single contaminants is the main approach to assess health risks, but epidemiological studies which investigate the relationships between exposure and health directly in the affected population have contributed important evidence. Limitations in exposure assessment have substantially contributed to uncertainty about associations found in epidemiological studies. OBJECTIVES: to examine exposure assessment methods that have been used in epidemiological studies on ICSs and to provide recommendations for improved exposure assessment in epidemiological studies by comparing exposure assessment methods in epidemiological studies and risk assessments. METHODS: after defining the multi-media framework of exposure related to ICSs, we discussed selected multi-media models applied in Europe. We provided an overview of exposure assessment in 54 epidemiological studies from a systematic review of hazardous waste sites; a systematic review of 41 epidemiological studies on incinerators and 52 additional studies on ICSs and health identified for this review. RESULTS: we identified 10 multi-media models used in Europe primarily for risk assessment. Recent models incorporated estimation of internal biomarker levels. Predictions of the models differ particularly for the routes ‘indoor air inhalation’ and ‘vegetable consumption’. Virtually all of the 54 hazardous waste studies used proximity indicators of exposure, based on municipality or zip code of residence (28 studies) or distance to a contaminated site (25 studies). One study used human biomonitoring. In virtually all epidemiological studies, actual land use was ignored. In the 52 additional studies on contaminated sites, proximity indicators were applied in 39 studies, air pollution dispersion modelling in 6 studies, and human biomonitoring in 9 studies. Exposure assessment in epidemiological studies on incinerators included indicators (presence of source in municipality and distance to the incinerator) and air dispersion modelling. Environmental multi-media modelling methods were not applied in any of the three groups of studies. CONCLUSIONS: recommendations for refined exposure assessment in epidemiological studies included the use of more sophisticated exposure metrics instead of simple proximity indicators where feasible, as distance from a source results in misclassification of exposure as it ignores key determinants of environmental fate and transport, source characteristics, land use, and human consumption behaviour. More validation studies using personal exposure or human biomonitoring are needed to assess misclassification of exposure. Exposure assessment should take more advantage of the detailed multi-media exposure assessment procedures developed for risk assessment. The use of indicators can be substantially improved by linking definition of zones of exposure to existing knowledge of extent of dispersion. Studies should incorporate more often land use and individual behaviour

Viral epidemics in a cell culture: novel high resolution data and their interpretation by a percolation theory based model

Because of its relevance to everyday life, the spreading of viral infections has been of central interest in a variety of scientific communities involved in fighting, preventing and theoretically interpreting epidemic processes. Recent large scale observations have resulted in major discoveries concerning the overall features of the spreading process in systems with highly mobile susceptible units, but virtually no data are available about observations of infection spreading for a very large number of immobile units. Here we present the first detailed quantitative documentation of percolation-type viral epidemics in a highly reproducible in vitro system consisting of tens of thousands of virtually motionless cells. We use a confluent astroglial monolayer in a Petri dish and induce productive infection in a limited number of cells with a genetically modified herpesvirus strain. This approach allows extreme high resolution tracking of the spatio-temporal development of the epidemic. We show that a simple model is capable of reproducing the basic features of our observations, i.e., the observed behaviour is likely to be applicable to many different kinds of systems. Statistical physics inspired approaches to our data, such as fractal dimension of the infected clusters as well as their size distribution, seem to fit into a percolation theory based interpretation. We suggest that our observations may be used to model epidemics in more complex systems, which are difficult to study in isolation.Comment: To appear in PLoS ONE. Supporting material can be downloaded from http://amur.elte.hu/BDGVirus

Elemental maps from EFTEM images using two different background subtraction models.

Acquisition of a great number of energy-filtered images in a TEM (EFTEM) around the characteristic signal with a low energy-selecting slit allows display of the electron energy loss (EEL)-spectrum of regions of interest (ROIs) of a sample. These EEL-spectra can be submitted to the different treatments already in use for electron energy loss spectroscopy (EELS). In particular, it is possible to fit the experimental background with different mathematical models, using images acquired below and above a characteristic ionization edge. After this fitting, elemental maps can be computed by subtraction of the extrapolated/interpolated background from the characteristic images. In this work, we compared two mathematical models for background fitting-the Egerton power law and the log-polynomial law. We studied the low-energy region (40-150 eV) and a higher-energy region (350-600 eV) with the aid of software for interactive processing of EFTEM image series that we developed. The analyzed elements were the constitutive elements: iron, phosphorus, nitrogen, and oxygen in several biological materials. Two analytical TEMs, one equipped with a post-column and the other with an in-column spectrometer, were used. Our experimental results confirm that the power law is very sensitive to the value of the energy loss of the pre-edge images when the background is computed by extrapolation. The log-polynomial model is less sensitive than the power law model to the value of the energy loss of the pre-edge images in the low energy region. For the oxygen K edge at 535 eV, it gives the best fit when it is combined with the interpolation method. The use of programs that facilitate the handling of EFTEM image series, and the controlled calculation of the background under the characteristic images, represent a step forward in the generation of elemental maps

Efficient computation of two-electron reduced density matrices via selected configuration interaction

We create an approach to efficiently calculate two-electron reduced density matrices (2-RDMs) using selected configuration interaction wavefunctions. This is demonstrated using the specific example of Monte Carlo configuration interaction (MCCI). The computation of the 2-RDMs is accelerated by using ideas from fast implementations of full configuration interaction (FCI) and recent advances in implementing the Slater-Condon rules using hardware bitwise operations. This method enables a comparison of MCCI and truncated CI 2-RDMs with FCI values for a range of molecules, which includes stretched bonds and excited states. The accuracy in energies, wavefunctions, and 2-RDMs is seen to exhibit a similar behavior. We find that MCCI can reach sufficient accuracy of the 2-RDM using significantly fewer configurations than truncated CI, particularly for systems with strong multireference character

Towards high-resolution X-ray scattering as a probe of electron correlation

X-ray scattering cross sections are calculated using a range of increasingly correlated methods: Hartree&ndash;Fock (HF), complete active space self-consistent field (CASSCF), Monte Carlo configuration interaction (MCCI), and full configuration interaction (FCI). Even for the seemingly straightforward case of ground state Ne, the accuracy of the total scattering is significantly better with a more correlated wavefunction. Scanning the bond distance in ground state CO shows that the total scattering signal tracks the multireference character. We examine the convergence of the elastic, inelastic, and total scattering of O3. Overall, the inelastic and total components are found to be the most sensitive to the strength of correlation. Our results suggest that highly accurate measurement of X-ray scattering could provide a sensitive probe of pair-wise correlation between electrons.</p