Obesity is highly co-morbid with anxiety and/or depression in children, conditions that may further worsen the metabolic and cardiovascular risks for obese individuals. Dysregulation of the hypothalamic-pituitary-adrenal axis is involved in the pathophysiology of anxiety disorders, depression, and obesity, and diverse cortisol concentrations may be found in obese children, depending on their degree of psychological distress. The aim of this study was to examine cortisol profiles among obese children with or without symptoms of anxiety and depression. A group of 128 children (53% females; mean age ± SD: 11.2 ± 2.2 years) derived from a pediatric obesity clinic were studied. Anxiety and depressive symptomatology were assessed with appropriate instruments. Morning serum and five diurnal salivary cortisol concentrations were measured. Obese children were 3.1/2.3 times more likely to report state and trait anxiety, respectively, and 3.6 times more likely to report depressive symptoms than children of the same age group, from a contemporary Greek sample. Trait anxiety and noon salivary cortisol concentrations were significantly positively correlated (p = 0.002). Overall, salivary cortisol concentrations were increased in children with anxiety or depression symptomatology compared to obese children without any affective morbidity (p = 0.02) and to those with anxiety and depression co-morbidity (p = 0.02). In conclusion, in obese children, emotional distress expressed by symptoms of anxiety and/or depression is associated with circadian cortisol profiles reflecting a potential pathway for further morbidity. Longitudinal studies may reveal a role of cortisol in linking obesity, anxiety, and depression to the development of further psychological and physical morbidity.

The sensitivity of aequorin-based bioluminometric hybridization assays was enhanced by introducing, enzymically, multiple aequorin labels per DNA hybrid. The target DNA was hybridized in microtiter wells with an immobilized capture probe and a digoxigenin-labeled detection probe. The hybrids were reacted with an anti-digoxigenin antibody conjugated to horseradish peroxidase. Peroxidase catalyzed the oxidation of digoxigenin-tyramine by hydrogen peroxide, resulting in the attachment of multiple digoxigenin moieties to the solid phase. Aequorin-labeled anti-digoxigenin antibody was then allowed to bind to the immobilized digoxigenins. The bound aequorin was determined by its characteristic Ca2+-triggered bioluminescence. As low as 20 fmol/L (1 amol/ well) target DNA was detected with a signal-to-background ratio of 2.7. A hybridization assay that used only aequorin-labeled anti-digoxigenin antibody without the peroxidase amplification step gave a signal-to-background ratio of 2 for 160 fmol/L target DNA. The signal enhancement of the amplified assay was in the range of 14-38 times. The analytical range of the amplified assay extended up to 2600 fmol/L. The CVs were in the range of 5.5-7.3%.

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Two cDNAs encoding firefly luciferase (FLuc) and Renilla luciferase (RLuc) were used as labels for the development of a microtiter well-based expression hybridization assay that allows simultaneous determination of two target DNA sequences. The target DNAs were denatured and hybridized with specific capture and detection probes. One detection probe was biotinylated while the other was tailed with poly(dT). The hybrids were reacted with a streptavidin-FLuc DNA complex and a poly(dA)-tailed RLuc DNA, respectively. Subsequently, the cDNA labels were expressed in vitro simultaneously and independently in the same transcription/translation reaction mixture. The activities of generated firefly and Renilla luciferases were co-determined in the same sample based on the differential requirements of their characteristic bioluminescent reactions for magnesium ions.

In recent years an increasing amount of interest has been directed at the study and routine testing of polymorphisms responsible for variations in drug metabolism. Most of the current methods involve either time-consuming electrophoresis steps or specialized and expensive equipment. In this context, we have developed a rapid, simple and robust method for genotyping of CYP2D6*3, CYP2D6*4, CYP2C19*2, CYP2C19*3 and TPMT*2 single nucleotide polymorphisms (SNP). Genomic DNA is isolated from whole blood and the segments that span the SNP of interest are amplified by PCR. The products are subjected directly (without purification) to two primer extension (PEXT) reactions (three cycles each) using normal and mutant primers in the presence of biotin-dUTP. The PEXT primers contain a (dA)(30) segment at the 5' end. The PEXT products are detected visually by a dry-reagent dipstick-type assay in which the biotinylated extension products are captured from immobilized streptavidin on the test zone of the strip and detected by hybridization with oligo(dT)-functionalized gold nanoparticles. Patient samples (76 variants in total) were genotyped and the results were fully concordant with those obtained by direct DNA sequencing.