Altered gray matter organization in children and adolescents with ADHD: a structural covariance connectome study.

Although multiple studies have reported structural deficits in multiple brain regions in attention-deficit hyperactivity disorder (ADHD), we do not yet know if these deficits reflect a more systematic disruption to the anatomical organization of large-scale brain networks. Here we used a graph theoretical approach to quantify anatomical organization in children and adolescents with ADHD. We generated anatomical networks based on covariance of gray matter volumes from 92 regions across the brain in children and adolescents with ADHD (n=34) and age- and sex-matched healthy controls (n=28). Using graph theory, we computed metrics that characterize both the global organization of anatomical networks (interconnectivity (clustering), integration (path length) and balance of global integration and localized segregation (small-worldness)) and their local nodal measures (participation (degree) and interaction (betweenness) within a network). Relative to Controls, ADHD participants exhibited altered global organization reflected in more clustering or network segregation. Locally, nodal degree and betweenness were increased in the subcortical amygdalae in ADHD, but reduced in cortical nodes in the anterior cingulate, posterior cingulate, mid temporal pole and rolandic operculum. In ADHD, anatomical networks were disrupted and reflected an emphasis on subcortical local connections centered around the amygdala, at the expense of cortical organization. Brains of children and adolescents with ADHD may be anatomically configured to respond impulsively to the automatic significance of stimulus input without having the neural organization to regulate and inhibit these responses. These findings provide a novel addition to our current understanding of the ADHD connectome.

Right external globus pallidus changes are associated with altered causal awareness in youth with depression.

Cognitive impairment is a functionally disabling feature of depression contributing to maladaptive decision-making, a loss of behavioral control and an increased disease burden. The ability to calculate the causal efficacy of ones actions in achieving specific goals is critical to normal decision-making and, in this study, we combined voxel-based morphometry (VBM), shape analysis and diffusion tensor tractography to investigate the relationship between cortical-basal ganglia structural integrity and such causal awareness in 43 young subjects with depression and 21 demographically similar healthy controls. Volumetric analysis determined a relationship between right pallidal size and sensitivity to the causal status of specific actions. More specifically, shape analysis identified dorsolateral surface vertices where an inward location was correlated with reduced levels of causal awareness. Probabilistic tractography revealed that affected parts of the pallidum were primarily connected with the striatum, dorsal thalamus and hippocampus. VBM did not reveal any whole-brain gray matter regions that correlated with causal awareness. We conclude that volumetric reduction within the indirect pathway involving the right dorsolateral pallidum is associated with reduced awareness of the causal efficacy of goal-directed actions in young depressed individuals. This causal awareness task allows for the identification of a functionally and biologically relevant subgroup to which more targeted cognitive interventions could be applied, potentially enhancing the long-term outcomes for these individuals.

Sequencing and expression of additional xylanase genes from the hyperthermophile Thermotoga maritima FjSS3B.1.

Two genes, xynB and xynC, coding for xylanases were isolated from Thermotoga maritima FjSS3B.1 by a genomic-walking-PCR technique. Sequencing of the genes showed that they encode multidomain family 10 xylanases. Only XynB exhibited activity against xylan substrates. The temperature optimum (87 degrees C) and pH optimum (pH 6.5) of XynB are different from the previously reported xylanase, XynA (also a family 10 enzyme), from this organism. The catalytic domain expressed without other domains has a lower temperature optimum, is less thermostable, and has optimal activity at pH 6.5. Despite having a high level of sequence similarity to xynB, xynC appears to be nonfunctional since its encoded protein did not show significant activity on xylan substrates.

Evaluation of fluorochromes and excitation sources for immunofluorescence in water samples.

Fluorescent labelling methods for detecting microorganisms in water have limited sensitivity partly due to the natural autofluorescence from environmental particles. The aim of this study was to examine the autofluorescence of water samples to determine the optimal excitation source and fluorescent labels for minimising background autofluorescence and therefore enhancing sensitive detection of Cryptosporidium oocysts. Particles concentrated from water were examined using fluorimetry at a wide range of excitation wavelengths to determine their autofluorescent properties. Two major peaks were identified emitting at 390 to 510 nm and at 640 to 700 nm. Flow cytometry was used to define the optical properties of oocysts immunofluorescently labelled with a range of fluorochromes. Concentrated water samples were analysed using flow cytometry and the number of particles with fluorescence and light scatter properties similar to the fluorescently labelled oocysts recorded. Fluorescein isothiocyanate exited at 488 nm was the most suitable label for oocysts in untreated water with less than 70 particles having optical properties similar to labelled oocysts, detected in 10 litre concentrates. The fluorochromes CY3, phycoerythrin (PE), and tetramethylrhodamine B thioisocyanate (TRITC) excited at 542 nm were the most suitable labels for oocysts in drinking water with less than 40 particles having optical properties similar to labelled oocysts, detected in 100 litre concentrates.

Simple and rapid measurement of Cryptosporidium excystation using flow cytometry.

In vitro excystation is commonly used to determine the viability of samples of purified Cryptosporidium parvum oocysts. Following exposure to conditions that stimulate excystation, samples are examined microscopically to determine the number of excysted oocysts. The microscopy procedure is tedious and time consuming, and difficult to apply to most oocyst samples without a purification step. A simple flow cytometric method was developed for determining the numbers of oocysts that had excysted following the in vitro excystation procedure. Differences in light-scatter properties were used to differentiate intact, partially empty and empty oocysts. By staining samples with a monoclonal antibody specific to the oocyst wall it was possible to apply the technique to unpurified oocysts from faeces. Correlation of the flow cytometric and microscopic method was statistically significant (P < 0.05), resulting in a calculated correlation coefficient of 0.994. The flow cytometry method is faster and more sensitive than the microscopy procedure, and enables analysis of large numbers of samples and of many thousands of oocysts in each sample.

Isolation of glycosylation mutants in Dictyostelium discoideum using flow cytometry.

Fluorescence-activated cell sorting was used to isolate five spores of the soil amoeba Dictyostelium discoideum that carried new glycosylation mutations, which were produced by restriction enzyme-mediated integration (REMI)-induced gene disruption and which occurred at frequencies of around 10(-5). These mutations were identified by the loss of an O-glycosylation epitope found on surface proteins of wild type D. discoideum spores that is recognised by the monoclonal antibody MUD62. A secondary antibody conjugated to the fluorochrome fluorescein isothiocyanate identified MUD62 bound to spores. Spores lacking this epitope did not fluoresce, allowing this population to be separated. Samples were found to contain around 0.1% of viable spores that were wild type but lacked the MUD62 epitope at the time of sorting. To remove these spores from the unlabelled population, samples were labelled with monoclonal antibody MUD50, which recognises surface proteins on immature spores and proteins exposed from an inner coat layer. Double labelling with MUD50 and MUD62 reduced the unlabelled viable population to less than 0.002%, allowing the glycosylation-defective spores to be isolated. This is the first use of a selective approach to isolate nonmorphological REMI-induced mutants in D. discoideum. This study also characterises the surface properties of spore types found in mature fruiting bodies of D. discoideum.