Background: Drosophila melanogaster females have two X chromosomes and two autosome sets (XX;AA), while males have a single X chromosome and two autosome sets (X;AA). Drosophila male somatic cells compensate for a single copy of the X chromosome by deploying male-specific-lethal (MSL) complexes that increase transcription from the X chromosome. Male germ cells lack MSL complexes, indicating that either germline X-chromosome dosage compensation is MSL-independent, or that germ cells do not carry out dosage compensation.

The cDNA microarray is one technological approach that has the potential to accurately measure changes in global mRNA expression levels. We report an assessment of an optimized cDNA microarray platform to generate accurate, precise and reliable data consistent with the objective of using microarrays as an acquisition platform to populate gene expression databases. The study design consisted of two independent evaluations with 70 arrays from two different manufactured lots and used three human tissue sources as samples: placenta, brain and heart. Overall signal response was linear over three orders of magnitude and the sensitivity for any element was estimated to be 2 pg mRNA. The calculated coefficient of variation for differential expression for all non-differentiated elements was 12-14% across the entire signal range and did not vary with array batch or tissue source. The minimum detectable fold change for differential expression was 1.4. Accuracy, in terms of bias (observed minus expected differential expression ratio), was less than 1 part in 10 000 for all non-differentiated elements. The results presented in this report demonstrate the reproducible performance of the cDNA microarray technology platform and the methods provide a useful framework for evaluating other technologies that monitor changes in global mRNA expression.

Further progress in the development of polymer gel dosimetry using MRI is reported, together with examples of its application to verify treatment plans for stereotactic radiosurgery and high dose rate brachytherapy. The dose distribution image produced in the tissue-equivalent gel by radiation-induced polymerization, and encoded in the spatial distribution of the NMR transverse relaxation rates (R2) of the water protons in the gel, is permanent. Maps of R2 are constructed from magnetic resonance imaging data and serve as a template for dose maps, which can be used to verify complex dose distributions from external sources or brachytherapy applicators. The integrating, three-dimensional, tissue-equivalent characteristics of polymer gels make it possible to obtain dose distributions not readily measured by conventional methods. An improved gel formulation (BANG-2) has a linear dose response that is independent of energy and dose rate for the situations studied to date. There is excellent agreement between the dose distributions predicted using treatment planning calculations and those measured using the gel method, and the clinical practical utility of MRI-based polymer gel dosimetry is thereby demonstrated.

A critical question in Alzheimer's disease (AD) research is the cause of memory loss that leads to dementia. The amyloid precursor protein + presenilin-1 (APP+PS1) transgenic mouse is a model for amyloid deposition, and like AD, the mice develop memory deficits as amyloid deposits accumulate. We profiled gene expression in these transgenic mice by microarray and quantitative RT-PCR (qRT-PCR). At the age when these animals developed cognitive dysfunction, they had reduced mRNA expression of several genes essential for long-term potentiation and memory formation (Arc, Zif268, NR2B, GluR1, Homer-1a, Nur77/TR3). These changes appeared to be related to amyloid deposition, because mRNA expression was unchanged in the regions that did not accumulate amyloid. Transgene expression was similar in both amyloid-containing and amyloid-free regions of the brain. Interestingly, these changes occurred without apparent changes in synaptic structure, because a number of presynaptic marker mRNAs (growth-associated protein-43, synapsin, synaptophysin, synaptopodin, synaptotagmin, syntaxin) remained stable. Additionally, a number of genes related to inflammation were elevated in transgenic mice, primarily in the regions containing amyloid. In AD cortical tissue, the same memory-associated genes were downregulated. However, all synaptic and neuronal transcripts were reduced, implying that the loss of neurons and synapses contributed to these changes. We conclude that reduced expression of selected genes associated with memory consolidation are linked to memory loss in both circumstances. This suggests that the memory loss in APP+PS1 transgenic mice may model the early memory dysfunction in AD before the degeneration of synapses and neurons.

Background: Sexual dimorphism results in the formation of two types of individuals with specialized reproductive roles and is most evident in the germ cells and gonads.

We report a quantum dot (Qdot) nanobarcode-based microbead random array platform for accurate and reproducible gene expression profiling in a high-throughput and multiplexed format. Four different sizes of Qdots, with emissions at 525, 545, 565, and 585 nm are mixed with a polymer and coated onto the 8-mum-diameter magnetic microbeads to generate a nanobarcoded bead termed as QBeads. Twelve intensity levels for each of the four colors were used. Gene-specific oligonucleotide probes are conjugated to the surface of each spectrally nanobarcoded bead to create a multiplexed panel, and biotinylated cRNAs are generated from sample total RNA and hybridized to the gene probes on the microbeads. A fifth streptavidin Qdot (655 nm or infrared Qdot) binds to biotin on the cRNA, acting as a quantification reporter. Target identity was decoded based on spectral profile and intensity ratios of the four coding Qdots (525, 545, 565, and 585 nm). The intensity of the 655 nm Qdot reflects the level of biotinylated cRNA captured on the beads and provides the quantification for the corresponding target gene. The system shows a sensitivity of < or =10(4) target molecules detectable with T7 amplification, a level that is better than the 10(5) number achievable with a high-density microarray system, and approaching the 10(3)-10(4) level usually observed for quantitative PCR (qPCR). The QBead nanobarcode system has a dynamic range of 3.5 logs, better than the 2-3 logs observed on various microarray platforms. The hybridization reaction is performed in liquid phase and completed in 1-2 hours, at least 1 order of magnitude faster than microarray-based hybridizations. Detectable fold change is lower than 1.4-fold, showing high precision even at close to single copy per cell level. Reproducibility for this proof-of-concept study approaches that of Affymetrix GeneChip microarray, with an R(2) value between two repeats at 0.984, and interwell CV around 5%. In addition, it provides increased flexibility, convenience, and cost-effectiveness in comparison to conventional gene expression profiling methods.

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Rheumatoid arthritis (RA) is a chronic inflammatory disorder that primarily involves the joints. Accurate and frequent assessment of RA disease activity is critical to optimal treatment planning. A novel algorithm has been developed to determine a multi-biomarker disease activity (MBDA) score based upon measurement of the concentrations of 12 serum biomarkers in multiplex format. Biomarker assays from several different platforms were used in feasibility studies to identify biomarkers of potential significance. These assays were adapted to a multiplex platform for training and validation of the algorithm. In this study, the analytical performance of the underlying biomarker assays and the MBDA score was evaluated. Quantification of 12 biomarkers was performed with multiplexed sandwich immunoassays in three panels. Biomarker-specific capture antibodies were bound to specific locations in each well; detection antibodies were labeled with electrochemiluminescent tags. Data were acquired with a Sector Imager 6000, and analyte concentrations were determined. Parallelism, dynamic range, cross-reactivity, and precision were established for each biomarker as well as for the MBDA score. Interference by serum proteins, heterophilic antibodies, and common RA therapies was also assessed. The individual biomarker assays had 3-4 orders of magnitude dynamic ranges, with good reproducibility across time, operators, and reagent lots; the MBDA score had a median coefficient of variation of <2% across the score range. Cross-reactivity as well as interference by serum rheumatoid factor (RF), human anti-mouse antibodies (HAMA), or common RA therapies, including disease-modifying antirheumatic drugs and biologics, was minimal. The same MBDA score was observed in different subjects despite having different biomarker profiles, supporting prior literature reports that multiple pathways contribute to RA.