Combined Fluorescent-Chromogenic In Situ Hybridization for Identification and Laser Microdissection of Interphase Chromosomes

Chromosome territories constitute the most conspicuous feature of nuclear architecture, and they exhibit non-random distribution patterns in the interphase nucleus. We observed that in cell nuclei from humans with Down Syndrome two chromosomes 21 frequently localize proximal to one another and distant from the third chromosome. To systematically investigate whether the proximally positioned chromosomes were always the same in all cells, we developed an approach consisting of sequential FISH and CISH combined with laser-microdissection of chromosomes from the interphase nucleus and followed by subsequent chromosome identification by microsatellite allele genotyping. This approach identified proximally positioned chromosomes from cultured cells, and the analysis showed that the identity of the chromosomes proximally positioned varies. However, the data suggest that there may be a tendency of the same chromosomes to be positioned close to each other in the interphase nucleus of trisomic cells. The protocol described here represents a powerful new method for genome analysis

Análisis del nivel de sedentarismo mediante acelerometría y cuestionario IPAQ en personas con hipertensión primaria y sobrepeso u obesidad

Antecedentes: La hipertensión arterial y el sobrepeso u obesidad son considerados factores de riesgo cardiovascular, asociados de forma clara con el sedentarismo en la población adulta. La valoración del sedentarismo se puede realizar a través de métodos objetivos y subjetivos. Objetivos: Analizar el nivel de sedentarismo, mediante el cuestionario internacional de actividad física (AF) (IPAQ, método subjetivo) y acelerometría (método objetivo) en personas con HTA primaria y sobrepeso u obesidad. Determinar si existen diferencias entre ambos métodos de valoración y entre sexos. Métodos: Se llevaron a cabo las dos pruebas de valoración a 48 participantes del estudio EXERDIET-HTA (edad 51,9 ± 8,1 años). 1) Cuestionario IPAQ en versión corta que mide la AF realizada durante los últimos siete días. Los datos se clasificaron en tiempo sedentario, ligero, moderado o intenso según las recomendaciones generales de la Organización Mundial de la Salud. 2) Acelerometría triaxial durante siete días portado en la muñeca no dominante. El tiempo dedicado a conductas sedentarias, así como las diferentes intensidades de AF (ligera, moderada, vigorosa y muy vigorosa), Kcal, METs y tiempo de sueño se calcularon para los días laborables y de fin de semana. Resultados: IPAQ presentó una media de 6 h/día en comportamientos sedentarios en ambos sexos. No se presentaron diferencias entre sexos. El 85,4% resulta sedentario. Acelerometría: Los participantes presentan 1,7±0,2 MET de media al día, considerado un valor metabólico de gasto calórico ligero. El tiempo de sueño diario fue de 6,6h. El gasto calórico diario se corresponde con comportamientos sedentarios entre semana para hombres y mujeres (2038,5±801,6 kcal) y sólo los hombres incrementan el gasto el fin de semana (P=0,036). Se observó que las mujeres realizan más minutos de AF a intensidad moderada que los hombres durante toda la semana (P=0,029). Se observaron diferencias de actividad entre los días entre semana y el fin de semana, con más tiempo de sedentarismo entre semana (P=0,040) y más AF ligera el fin de semana (P=0,003), resultando esta diferencia debida al cambio de actividad en los hombres pero no en las mujeres. Todos los participantes superan los 150 minutos de AF moderada que se recomienda en las guías mundiales según la acelerometría. Conclusiones: Las personas hipertensas y con sobrepeso u obesidad presentan en general conductas sedentarias. Los patrones de sueño podrían estar relacionados con la enfermedad. Sería necesario reducir las conductas sedentarias como prevención de la obesidad y del desarrollo prematuro de enfermedades cardiovasculares, fomentando la práctica regular de AF y mejorando los hábitos de alimentación.

Alcohol-Related Brain Damage in Humans

Chronic excessive alcohol intoxications evoke cumulative damage to tissues and organs. We examined prefrontal cortex (Brodmann's area (BA) 9) from 20 human alcoholics and 20 age, gender, and postmortem delay matched control subjects. H & E staining and light microscopy of prefrontal cortex tissue revealed a reduction in the levels of cytoskeleton surrounding the nuclei of cortical and subcortical neurons, and a disruption of subcortical neuron patterning in alcoholic subjects. BA 9 tissue homogenisation and one dimensional polyacrylamide gel electrophoresis (PAGE) proteomics of cytosolic proteins identified dramatic reductions in the protein levels of spectrin beta II, and alpha- and beta-tubulins in alcoholics, and these were validated and quantitated by Western blotting. We detected a significant increase in a-tubulin acetylation in alcoholics, a non-significant increase in isoaspartate protein damage, but a significant increase in protein isoaspartyl methyltransferase protein levels, the enzyme that triggers isoaspartate damage repair in vivo. There was also a significant reduction in proteasome activity in alcoholics. One dimensional PAGE of membrane-enriched fractions detected a reduction in beta-spectrin protein levels, and a significant increase in transmembranous alpha 3 (catalytic) subunit of the Na+, K+-ATPase in alcoholic subjects. However, control subjects retained stable oligomeric forms of a-subunit that were diminished in alcoholics. In alcoholics, significant loss of cytosolic alpha-and beta-tubulins were also seen in caudate nucleus, hippocampus and cerebellum, but to different levels, indicative of brain regional susceptibility to alcohol-related damage. Collectively, these protein changes provide a molecular basis for some of the neuronal and behavioural abnormalities attributed to alcoholics

Interaction between the 5-HT system and the basal ganglia: functional implication and therapeutic perspective in Parkinson's disease

The neurotransmitter serotonin (5-HT) has a multifaceted function in the modulation of information processing through the activation of multiple receptor families, including G-protein-coupled receptor subtypes (5-HT1, 5-HT2, 5-HT4-7) and ligand-gated ion channels (5-HT3). The largest population of serotonergic neurons is located in the midbrain, specifically in the raphe nuclei. Although the medial and dorsal raphe nucleus (DRN) share common projecting areas, in the basal ganglia (BG) nuclei serotonergic innervations come mainly from the DRN. The BG are a highly organized network of subcortical nuclei composed of the striatum (caudate and putamen), subthalamic nucleus (STN), internal and external globus pallidus (or entopeduncular nucleus in rodents, GPi/EP and GPe) and substantia nigra (pars compacta, SNc, and pars reticulata, SNr). The BG are part of the cortico-BG-thalamic circuits, which play a role in many functions like motor control, emotion, and cognition and are critically involved in diseases such as Parkinson's disease (PD). This review provides an overview of serotonergic modulation of the BG at the functional level and a discussion of how this interaction may be relevant to treating PD and the motor complications induced by chronic treatment with L-DOPA.

A New Method for Feedback on the Quality of Chest Compressions during Cardiopulmonary Resuscitation

Quality of cardiopulmonary resuscitation (CPR) improves through the use of CPR feedback devices. Most feedback devices integrate the acceleration twice to estimate compression depth. However, they use additional sensors or processing techniques to compensate for large displacement drifts caused by integration. This study introduces an accelerometer-based method that avoids integration by using spectral techniques on short duration acceleration intervals. We used a manikin placed on a hard surface, a sternal triaxial accelerometer, and a photoelectric distance sensor (gold standard). Twenty volunteers provided 60 s of continuous compressions to test various rates (80-140 min(-1)), depths (3-5 cm), and accelerometer misalignment conditions. A total of 320 records with 35312 compressions were analysed. The global root-mean-square errors in rate and depth were below 1.5 min(-1) and 2 mm for analysis intervals between 2 and 5 s. For 3 s analysis intervals the 95% levels of agreement between the method and the gold standard were within -1.64-1.67 min(-1) and -1.69-1.72 mm, respectively. Accurate feedback on chest compression rate and depth is feasible applying spectral techniques to the acceleration. The method avoids additional techniques to compensate for the integration displacement drift, improving accuracy, and simplifying current accelerometer-based devices.