Array biosensor based on enzyme kinetics monitoring by fluorescence spectroscopy: application for neurotoxins detection

The aim of the present work is to develop an evanescence wave array biosensor exploiting the “kinetic” approach of enzymatic reaction and further detection of the reaction products via pH sensitive fluorophore reporter. To demonstrate the feasibility of this approach, we have developed a biosensor array with the potential for direct detection of organophosphates using as a biorecognition element, an enzyme organophosphorus hydrolase (OPH), conjugated with a pH-sensitive fluorophore, carboxynaphthofluorescein (CNF). The presence of reference spots allows the discrimination of the enzymatic and non-enzymatic based pH changes; bovine serum albumin (BSA) was used as a non-enzymatic scaffold protein for CNF attachment at the reference spots. An array biosensor unit developed at the Naval Research Laboratories (NRL) was adopted as the detection platform and appropriately modified for enzyme-based measurements. A planar multi-mode waveguide was covered with an optically transparent TiO₂ layer to increase the surface area available for immobilization.

The biosensor enabled the detection of 2.5 μM paraoxon, and 10 μM DFP and parathion, respectively. Very short response time of 30 s can be achieved with a total analysis time of less than 2 min. When operated at room temperature and stored at 4 °C, the waveguide retained reasonable activity for greater than 45 days.     

Post-zygotic breakage of a dicentric chromosome results in mosaicism for a telocentric 9p marker chromosome in a boy with developmental delay

Chromosomal rearrangements resulting in an inverted duplication and a terminal deletion (inv dup del) can occur due to three known mechanisms, two of them resulting in a normal copy region between the duplicated regions. These mechanisms involve the formation of a dicentric chromosome, which undergo breakage during cell division resulting in cells with either an inverted duplication and deletion or a terminal deletion. We describe a mosaic 3 year old patient with two cell lines carrying a chromosome 9p deletion where one of the cell lines contains an additional telocentric marker chromosome. Our patient is mosaic for the product of a double breakage of a dicentric chromosome including a centric fission. Mosaicism involving different rearrangements of the same chromosome is rare and suggests an early mitotic breakage event.     

A novel combined 15q11.2 duplication and a bisatellited supernumerary marker derived from chromosome 22: Molecular characterization of the marker

Supernumerary marker chromosomes (SMC) are heterogeneous group of chromosomes which are reported in variable phenotypes. Approximately 70% originate from acrocentric chromosomes. Here we report a couple with recurrent miscarriages and a SMC originating from an acrocentric chromosome. The cytogenetic analysis of the husband revealed a karyotype of 47,XY+marker whereas the wife had a normal karyotype. Analysis of SMC with C-banding showed the presence of a big centromere in the center and silver staining showed prominent satellites on both sides of the marker. Apparently, microarray analysis revealed a 2.1 Mb duplication of 15q11.2 region but molecular cytogenetic analysis by fluorescence in situ hybridization (FISH) with whole chromosome paint (WCP) 15 showed that the SMC is not of chromosome 15 origin. Subsequently, FISH with centromere 22 identified the SMC to originate from chromosome 22 which was also confirmed by WCP 22. Additional dual FISH with centromere 22 and Acro-p-arm probes confirmed the centromere 22 and satellites on the SMC. Further fine mapping of the marker with Bacterial Artificial Chromosome (BAC) clones; two on chromosome 22 and four on chromosome 15 determined the marker to possess only centromere 22 sequences and that the duplication 15 exists directly on chromosome 15. In our study, we had identified and characterized a SMC showing inversion duplication 22(p11.1) combined with a direct tandem duplication of 15q11.2. The possible genotype–phenotype in relation with the two rearrangements is discussed.     

Detergent screening of a G-protein-coupled receptor using serial and array biosensor technologies

We describe the benefits and limitations of two biosensor approaches for screening solubilization conditions for G-protein-coupled receptors (GPCRs). Assays designed for a serial processing instrument (Biacore 2000/3000/T100) and an array platform (Biacore Flexchip) were used to examine how effectively 96 different detergents solubilized the chemokine receptor CCR5 while maintaining its binding activity for a conformationally sensitive Fab (2D7). Using the serial processing instrument, we were able to analyze three samples in each 30-min binding cycle, thereby requiring approximately 24 h to screen an entire 96-well plate of conditions. In-line capturing allowed us to normalize the 2D7 binding responses for different receptor capture levels. In contrast, with the array system, we could characterize the effects of all 96 detergents simultaneously, completing the assay in less than 1 h. But the current array technology requires that we capture the GPCR preparations off-line, making it more challenging to normalize for receptor capture levels. Also, the array platform is less sensitive than the serial platforms, thereby limiting the size of the analyte to larger molecules (>5000 Da). Overall, the two approaches proved to be highly complementary; both assays identified identical detergents that produced active solubilized CCR5 as well as those detergents that either were ineffective solubilizers or inactivated the receptor.     

The human blood DNA methylome displays a highly distinctive profile compared with other somatic tissues

In mammals, DNA methylation profiles vary substantially between tissues. Recent genome-scale studies report that blood displays a highly distinctive methylomic profile from other somatic tissues. In this study, we sought to understand why blood DNA methylation state is so different to the one found in other tissues. We found that whole blood contains approximately twice as many tissue-specific differentially methylated positions (tDMPs) than any other somatic tissue examined. Furthermore, a large subset of blood tDMPs showed much lower levels of methylation than tDMPs for other tissues. Surprisingly, these regions of low methylation in blood show no difference regarding genomic location, genomic content, evolutionary rates, or histone marks when compared to other tDMPs. Our results reveal why blood displays a distinctive methylation profile relative to other somatic tissues. In the future, it will be important to study how these blood specific tDMPs are mechanistically involved in blood-specific functions.     

Identification of Differential Aberrations in Multiple‐Sample Array CGH Studies

Summary Most existing methods for identifying aberrant regions with array CGH data are confined to a single target sample. Focusing on the comparison of multiple samples from two different groups, we develop a new penalized regression approach with a fused adaptive lasso penalty to accommodate the spatial dependence of the clones. The nonrandom aberrant genomic segments are determined by assessing the significance of the differences between neighboring clones and neighboring segments. The algorithm proposed in this article is a first attempt to simultaneously detect the common aberrant regions within each group, and the regions where the two groups differ in copy number changes. The simulation study suggests that the proposed procedure outperforms the commonly used single‐sample aberration detection methods for segmentation in terms of both false positives and false negatives. To further assess the value of the proposed method, we analyze a data set from a study that identified the aberrant genomic regions associated with grade subgroups of breast cancer tumors.     

Effect of Hydrotropism on Root System Development in Soybean ( Glycine max): Growth Experiments and a Model Simulation

To observe root system development, soybean plants (Glycine max) were grown in root boxes that were set horizontally to reduce the effect of gravity. Along with the root system development, the two-dimensional distribution of soil water content in the root boxes was measured continuously by the time domain reflectometry (TDR) method. Root system development and its morphological architecture were strongly affected by the positions of the water supply. It is suggested that root hydrotropism plays the dominant role in root system development. In addition to root hydrotropism, the importance of root compensatory growth is suggested. A combined model of root system development and soil water flow considering root hydrotropism and compensatory growth was used to simulate root system development and soil water flow. The morphological architecture of root systems and the distribution of soil water content obtained in the experiment were successfully explained by the model simulation. These results confirmed that root hydrotropism and compensatory growth are dominant factors in root system development under a reduced effect of gravity. The validity of the model was confirmed, and its applications for various purposes were suggested.     

iEMG: Imaging electromyography

Advanced data analysis and visualization methodologies have played an important role in making surface electromyography both a valuable diagnostic methodology of neuromuscular disorders and a robust brain–machine interface, usable as a simple interface for prosthesis control, arm movement analysis, stiffness control, gait analysis, etc. But for diagnostic purposes, as well as for interfaces where the activation of single muscles is of interest, surface EMG suffers from severe crosstalk between deep and superficial muscle activation, making the reliable detection of the source of the signal, as well as reliable quantification of deeper muscle activation, prohibitively difficult. To address these issues we present a novel approach for processing surface electromyographic data. Our approach enables the reconstruction of 3D muscular activity location, making the depth of muscular activity directly visible. This is even possible when deep muscles are overlaid with superficial muscles, such as seen in the human forearm. The method, which we call imaging EMG (iEMG), is based on using the crosstalk between a sufficiently large number of surface electromyographic electrodes to reconstruct the 3D generating electrical potential distribution within a given area. Our results are validated by in vivo measurements of iEMG and ultrasound on the human forearm.